Prostate-specific membrane antigen and uses thereof

ABSTRACT

This invention provides an isolated mammalian nucleic acid molecule encoding an alternatively spliced prostate-specific membrane (PSM′) antigen. This invention provides an isolated nucleic acid molecule encoding a prostate-specific membrane antigen promoter. This invention provides a method of detecting hematogenous micrometastic tumor cells of a subject, and determining prostate cancer progression in a subject.

This application is a continuation-in-part of U.S. applications Ser.Nos. 08/466,381 and 08/470,735 both filed Jun. 6, 1995, and is acontinuations of U.S. Ser. No. 08/394,152, filed Feb. 24, 1995, now U.S.Pat. No. 5,935,818 the contents of which are hereby incorporated byreference.

This invention disclosed herein was made in part with Government supportunder NIH Grants No. DK47650 and CA58192, CA-39203, CA-29502,CA-08748-29 from the Department of Health and Human Services.Accordingly, the U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofeach set of Examples in the Experimental Details section.

Prostate cancer is among the most significant medical problems in theUnited States, as the disease is now the most common malignancydiagnosed in American males. In 1992 there were over 132,000 new casesof prostate cancer detected with over 36,000 deaths attributable to thedisease, representing a 17.3% increase over 4 years (2). Five yearsurvival rates for patients with prostate cancer range from 88% forthose with localized disease to 29% for those with metastatic disease.The rapid increase in the number of cases appears to result in part froman increase in disease awareness as well as the widespread use ofclinical markers such as the secreted proteins prostate-specific antigen(PSA) and prostatic acid phosphatase (PAP) (37).

The prostate gland is a site of significant pathology affected byconditions such as benign growth (BPH), neoplasia (prostatic cancer) andinfection (prostatitis). Prostate cancer represents the second leadingcause of death from cancer in man (1). However prostatic cancer is theleading site for cancer development in men. The difference between thesetwo facts relates to prostatic cancer occurring with increasingfrequency as men age, especially in the ages beyond 60 at a time whendeath from other factors often intervenes. Also, the spectrum ofbiologic aggressiveness of prostatic cancer is great,-so that in somemen following detection the tumor remains a latent histologic tumor anddoes not become clinically significant, whereas in other it progressesrapidly, metastasizes and kills the man in a relatively short 2-5 yearperiod (1, 3).

In prostate cancer cells, two specific proteins that are made in veryhigh concentrations are prostatic acid phosphatase (PAP) and prostatespecific antigen (PSA) (4, 5, 6). These proteins have been characterizedand have been used to follow response to therapy. With the developmentof cancer, the normal architecture of the gland becomes altered,including loss of the normal duct structure for the removal ofsecretions and thus the secretions reach the serum. Indeed measurementof serum PSA is suggested as a potential screening method for prostaticcancer. Indeed, the relative amount of PSA and/or PAP in the cancerreduces as compared to normal or benign tissue.

PAP was one of the earliest serum markers for detecting metastaticspread (4). PAP hydrolyses tyrosine phosphate and has a broad substratespecificity. Tyrosine phosphorylation is often increased with oncogenictransformation. It has been hypothesized that during neoplastictransformation there is less phosphatase activity available toinactivate proteins that are activated by phosphorylation on tyrosineresidues. In some instances, insertion of phosphatases that havetyrosine phosphatase activity has reversed the malignant phenotype.

PSA is a protease and it is not readily appreciated how loss of itsactivity correlates with cancer development (5, 6). The proteolyticactivity of PSA is inhibited by zinc. Zinc concentrations are high inthe normal prostate and reduced in prostatic cancer. Possibly the lossof zinc allows for increased proteolytic activity by PSA. As proteasesare involved in metastasis and some proteases stimulate mitoticactivity, the potentially increased activity of PSA could behypothesized to play a role in the tumors metastases and spread (7).

Both PSA and PAP are found in prostatic secretions. Both appear to bedependent on the presence of androgens for their production and aresubstantially reduced following androgen deprivation.

Prostate-specific membrane antigen (PSM) which appears to be localizedto the prostatic membrane has been identified. This antigen wasidentified as the result of generating monoclonal antibodies to aprostatic cancer cell, LNCaP (8).

Dr. Horoszewicz established a cell line designated LNCaP from the lymphnode of a hormone refractory, heavily pretreated patient (9). This linewas found to have an aneuploid human male karyotype. It maintainedprostatic differentiation functionality in that it produced both PSA andPAP. It possessed an androgen receptor of high affinity and specificity.Mice were immunized with LNCaP cells and hybridomas were derived fromsensitized animals. A monoclonal antibody was derived and was designated7E11-C5 (8). The antibody staining was consistent with a membranelocation and isolated fractions of LNCaP cell membranes exhibited astrongly positive reaction with immunoblotting and ELISA techniques.This antibody did not inhibit or enhance the growth of LNCaP cells invitro or in vivo. The antibody to this antigen was remarkably specificto prostatic epithelial cells, as no reactivity was observed in anyother component. Immunohistochemical staining of cancerous epithelialcells was more intense than that of normal or benign epithelial cells.

Dr. Horoszewicz also reported detection of immunoreactive material using7E11-C5 in serum of prostatic cancer patients (8). The immunoreactivitywas detectable in nearly 60% of patients with stage D-2 disease and in aslightly lower percentage of patients with earlier stage disease, butthe numbers of patients in the latter group are small. Patients withbenign prostatic hyperplasia (BPH) were negative. Patients with noapparent disease were negative, but 50-60% of patients in remission yetwith active stable disease or with progression demonstrated positiveserum reactivity. Patients with non prostatic tumors did not showimmunoreactivity with 7E11-C5.

The 7E11-C5 monoclonal antibody is currently in clinical trials. Thealdehyde groups of the antibody were oxidized and the linker-chelatorglycol-tyrosyl-(n, ε-diethylenetriamine-pentacetic acid)-lysine(GYK-DTPA) was coupled to the reactive aldehydes of the heavy chain(10). The resulting antibody was designated CYT-356. Immunohistochemicalstaining patterns were similar except that the CYT-356 modified antibodystained skeletal muscle. The comparison of CYT-356 with 7E11-C5monoclonal antibody suggested both had binding to type 2 muscle fibers.The reason for the discrepancy with the earlier study, which reportedskeletal muscle to be negative, was suggested to be due to differencesin tissue fixation techniques. Still, the most intense and definitereaction was observed with prostatic epithelial cells, especiallycancerous cells. Reactivity with mouse skeletal muscle was detected withimmunohistochemistry but not in imaging studies. The Indium¹¹¹-labeledantibody localized to LNCaP tumors grown in nude mice with an uptake ofnearly 30% of the injected dose per gram tumor at four days. In-vivo, noselective retention of the antibody was observed in antigen negativetumors such as PC-3 and DU-145, or by skeletal muscle. Very little wasknown about the PSM antigen. An effort at purification andcharacterization has been described at meetings by Dr. George Wright andcolleagues (11, 12).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Signal in lane 2 represent the 100 kD PSM antigen. The EGFr wasused as the positive control and is shown in lane 1. Incubation withrabbit antimouse (RAM) antibody alone served as negative control and isshown in lane 3.

FIGS. 2A-2D: Upper two photos show LNCaP cytospins staining positivelyfor PSM antigen. Lower left in DU-145 and lower right is PC-3 cytospin,both negative for PSM antigen expression.

FIGS. 3A-3D: Upper two panels are human prostate sections (BPH) stainingpositively for PSM antigen. The lower two panels show invasive prostatecarcinoma human sections staining positively for expression of the PSMantigen.

FIG. 4: 100 kD PSM antigen following immunoprecipitation of³⁵S-Methionine labelled LNCaP cells with Cyt-356 antibody.

FIG. 5: 3% agarose gels stained with Ethidium bromide revealing PCRproducts obtained using the degenerate PSM antigen primers. The arrowpoints to sample IN-20, which is a 1.1 kb PCR product which was laterconfirmed to be a partial cDNA coding for the PSM gene.

FIGS. 6A-6B: 2% agarose gels of plasmid DNA resulting from TA cloning ofPCR products. Inserts are excised from the PCR II vector (InvitrogenCorp.) by digestion with EcoRI. 1.1 kb PSM gene partial cDNA product isshown in lane 3 of gel 1.

FIG. 7: Autoradiogram showing size of cDNA represented in applicants'LNCaP library using M-MLV reverse transcriptase.

FIG. 8: Restriction analysis of full-length clones of PSM gene obtainedafter screening cDNA library. Samples have been cut with Not I and Sal Irestriction enzymes to liberate the insert.

FIG. 9: Plasmid Southern autoradiogram of full length PSM gene clones.Size is approximately 2.7 kb.

FIG. 10: Northern blot revealing PSM expression limited to LNCaPprostate cancer line and H26 Ras-transfected LNCaP cell line. PC-3,DU-145, T-24, SKRC-27, HELA, MCF-7, HL-60, and others were are allnegative.

FIG. 11: Autoradiogram of Northern analysis revealing expression of 2.8kb PSM message unique to the LNCaP cell line (lane 1), and absent fromthe DU-145 (lane 2) and PC-3 cell lines (lane 3). RNA size ladder isshown on the left (kb) and 28S and 18S ribosomal RNA bands are indicatedon the right.

FIGS. 12A-12B: Results of PCR of human prostate tissues using PSM geneprimers. Lanes are numbered from left to right. Lane 1, LNCaP; Lane 2,H26; Lane 3, DU-145; Lane 4, Normal Prostate; Lane 5, BPH; Lane 6,Prostate Cancer; Lane 7, BPH; Lane 8, Normal; Lane 9, BPH; Lane 10, BPH;Lane 11, BPH; Lane 12, Normal; Lane 13, Normal; Lane 14, Cancer; Lane15, Cancer; Lane 16, Cancer; Lane 17, Normal; Lane 18, Cancer; Lane 19,IN-20 Control; Lane 20, PSM cDNA

FIG. 13: Isoelectric point of PSM antigen (non-glycosylated)

FIGS. 14-A to 14-H: Secondary structure of PSM antigen

FIGS. 15A-15B: A. Hydrophilicity plot of PSM antigen (SEQ ID NO:128) B.Prediction of membrane spanning segments

FIGS. 16-A to 16-K: Homology with chicken, rat and human transferrinreceptor sequence. (SEQ ID NO:34-36)

FIGS. 17A-17C: Immunohistochemical detection of PSM antigen expressionin prostate cell lines. Top panel reveals uniformly high level ofexpression in LNCaP cells; middle panel and lower panel are DU-145 andPC-3 cells respectively, both negative.

FIG. 18: Autoradiogram of protein gel revealing products of PSM coupledin-vitro transcription/translation. Non-glycosylated PSM polypeptide isseen at 84 kDa (lane 1) and PSM glycoprotein synthesized following theaddition of microsomes is seen at 100 kDa (lane 2)

FIG. 19: Western Blot analysis detecting PSM expression in transfectednon-PSM expressing PC-3 cells. 100 kDa PSM glycoprotein species isclearly seen in LNCaP membranes (lane 1), LNCaP crude lysate (lane 2),and PSM-transfected PC-3 cells (lane 4), but is undetectable in nativePC-3 cells (lane 3).

FIG. 20: Autoradiogram of ribonuclease protection gel assaying for PSMmRNA expression in normal human tissues. Radiolabeled 1 kb DNA ladder(Gibco-BRL) is shown in lane 1. Undigested probe is 400 nucleotides(lane 2), expected protected PSM band is 350 nucleotides, and tRNAcontrol is shown (lane 3). A strong signal is seen in human prostate(lane 11), with very faint, but detectable signals seen in human brain(lane 4) and human salivary gland (lane 12).

FIG. 21: Autoradiogram of ribonuclease protection gel assaying for PSMmRNA expression in LNCaP tumors grown in nude mice, and in humanprostatic tissues. ³²P-labeled 1 kb DNA ladder is shown in lane 1. 298nucleotide undigested probe is shown (lane 2), and tRNA control is shown(lane 3). PSM mRNA expression is clearly detectable in LNCaP cells (lane4), orthotopically grown LNCaP tumors in nude mice with and withoutmatrigel (lanes 5 and 6), and subcutaneously implanted and grown LNCaPtumors in nude mice (lane 7). PSM mRNA expression is also seen in normalhuman prostate (lane 8), and in a moderately differentiated humanprostatic adenocarcinoma (lane 10). Very faint expression is seen in asample of human prostate tissue with benign hyperplasia (lane 9).

FIG. 22: Ribonuclease protection assay for PSM expression in LNCaP cellstreated with physiologic doses of various steroids for 24 hours.³²P-labeled DNA ladder is shown in lane 1. 298 nucleotide undigestedprobe is shown (lane 2), and tRNA control is shown (lane 3). PSM mRNAexpression is highest in untreated LNCaP cells in charcoal-strippedmedia (lane 4). Applicant see significantly diminished PSM expression inLNCaP cells treated with DHT (lane 5), Testosterone (lane 6), Estradiol(lane 7), and Progesterone (lane 8), with little response toDexamethasone (lane 9).

FIG. 23: Data illustrating results of PSM DNA and RNA presence intransfect Dunning cell lines employing Southern and Northern blottingtechniques

FIGS. 24A-24B: FIG. A indicates the power of cytokine transfected cellsto teach unmodified cells. Administration was directed to the parentalflank or prostate cells. The results indicate the microenvironmentconsiderations. FIG. B indicates actual potency at a particular site.The tumor was implanted in prostate cells and treated with immune cellsat two different sites.

FIGS. 25A-25B: Relates potency of cytokines in inhibiting growth ofprimary tumors. Animals administered un-modified parental tumor cellsand administered as a vaccine transfected cells. Following prostatectomyof rodent tumor results in survival increase.

FIG. 26: PCR amplification with nested primers improved the level ofdetection of prostatic cells from approximately one prostatic cell per10,000 MCF-7 cells to better than one cell per million MCF-7 cells,using either PSA.

FIG. 27: PCR amplification with nested primers improved the level ofdetection of prostatic cells from approximately one prostatic cell per10,000 MCF-7 cells to better than one cell per million MCF-7 cells,using PSM-derived primers.

FIG. 28: A representative ethidium stained gel photograph for PSM-PCR.Samples run in lane A represent PCR products generated from the outerprimers and samples in lanes labeled B are products of inner primerpairs.

FIG. 29: PSM Southern blot autoradiograph. The sensitivity of theSouthern blot analysis exceeded that of ethidium staining, as can beseen in several samples where the outer product is not visible on FIG.3, but is detectable by Southern blotting as shown in FIG. 4.

FIG. 30: Characteristics of the 16 patients analyzed with respect totheir clinical stage, treatment, serum PSA and PAP values, and resultsof assay.

FIGS. 31A-31D: The DNA sequence of the 3 kb XhoI fragment of p683 whichincludes 500 bp of DNA from the RNA start site was determined Sequence683XFRVS starts from the 5′ distal end of PSM promoter(SEQ ID NO: 109).

FIG. 32: Potential binding sites on the PSM promoter.

FIG. 33: Promoter activity of PSM up-stream fragment/CAT gene chimera.

FIG. 34: Comparison between PSM (SEQ ID NO:1) and PSM′ cDNA. (SEQ IDNO:1, nucleotides 1-112 and 381-2653). Sequence of the 5′ end of PSMcDNA (5) is shown. Underlined region denotes nucleotides which arepresent in PSM cDNA sequence but absent in PSM′ cDNA. Boxed regionrepresents the putative transmembrane domain of PSM antigen. * Asteriskdenotes the putative translation initiation site for PSM′.

FIG. 35: Graphical representation of PSM and PSM′ cDNA sequences andantisense PSM RNA probe (b). PSM cDNA sequence with complete codingregion (5) (a). PSM′ cDNA sequence from this study (c). Cross hatchedand open boxes denote sequences identity in PSM and PSM′. Hatched boxindicates sequence absent from PSM′. Regions of cDNA sequencecomplementary to the RNA probe are indicated by dashed lines between thesequences.

FIG. 36: RNase protection assay with PSM specific probe in primaryprostatic tissues. Total cellular RNA was isolated from human prostaticsamples: normal prostate, BPH, and CaP. PSM and PSM′ spliced variantsare indicated with arrows at right. The left lane is a DNA ladder.Samples from different patients are classified as: lanes 3-6, CaP,carcinoma of prostate; BPH, benign prostatic hypertrophy, lanes 7-9;normal, normal prostatic tissue, lanes 10-12. Autoradiograph was exposedfor longer period to read lanes 5 and 9.

FIG. 37: Tumor Index, a quantification of the expression of PSM andPSM′. Expression of PSM and PSM′ (FIG. 3) was quantified by densitometryand expressed as a ratio of PSM/PSM′ on the Y-axis. Three samples eachwere quantitated for primary CaP, BPH and normal prostate tissues. Twosamples were quantitated for LNCaP. Normal, normal prostate tissue.

FIG. 38: Characterization of PSM membrane bound and PSM′ in the cytosol.

FIG. 39: Intron 1F: Forward Sequence. Intron 1 contains a number oftrinucleotide repeats which can be area associated with chromosomalinstability in tumor cells. LNCaP cells and primary prostate tissue areidentical, however in the PC-3 and Du-145 tumors they have substantiallyaltered levels of these trinucleotide repeats which may relate to theirlack of expression of PSM (SEQ ID NO: 110).

FIGS. 40A-40B: Intron 1R: Reverse Sequence (SEQ ID NO: 111).

FIG. 41: Intron 2F: Forward Sequence (SEQ ID NO: 112).

FIG. 42 Intron 2R: Reverse Sequence (SEQ ID NO: 113).

FIGS. 43A-43B: Intron 3F: Forward Sequence (SEQ ID NO: 114).

FIGS. 44A-44B: Intron 3R: Reverse Sequence (SEQ ID NO: 115)

FIGS. 45A-45B: Intron 4F: Forward Sequence (SEQ ID NO: 116).

FIGS. 46A-46B: 4R: Reverse Sequence (SEQ ID NO: 117).

FIGS. 47A-47D: Sequence of the genomic region upstream of the 5′transcription start site of PSM (SEQ ID NO: 118).

FIG. 48: Photograph of ethidium bromide stained gel depictingrepresentative negative and positive controls used in the study. Samples1-5 were from, respectively: male with prostatis, a healthy femalevolunteer, a male with BPH, a control 1:1,000,000 dilution of LNCaPcells, and a patient with renal cell carcinoma. Below each reaction isthe corresponding control reaction performed with beta-2-microglobulinprimers to assure RNA integrity. No PCR products were detected for anyof these negative controls.

FIG. 49: Photograph of gel displaying representative positive PCRresults using PSM primers in selected patients with either localized ordisseminated prostate cancer. Sample 1-5 were from. respectively: apatient with clinically localized stage T1_(c) disease, a radicalprostatectomy patient with organ confined disease and a negative serumPSA, a radical prostatectomy patient with locally advanced disease and anegative serum PSA, a patient with treated stage D2 disease, and apatient with treated hormone refractory disease.

FIG. 50: Chromosomal location of PSM based on cosmid construction.

FIG. 51: Human monochromosomal somatic cell hybrid blot showing thatchromosome 11 contained the PSM genetic sequence by Southern analysis.DNA panel digested with PstI restriction enzyme and probed with PSMcDNA. Lanes M and H refer to mouse and hamster DNAs. The numberscorrespond to the human chromosomal DNA in that hybrid.

FIG. 52: Ribonuclease protection assay using PSM radiolabeled RNA proberevels an abundant PSM mRNA expression in AT6.1-11 clone 1, but not inAT6.1-11 clone 2, thereby mapping PSM to 11p11.2-13 region.

FIG. 53: Tissue specific expression of PSM RNA by Northern blotting andRNAse protection assay.

FIG. 54: Mapping of the PSM gene to the 11p11.2-p13 region of humanchromosome 11 by southern blotting and in-situ hybridization.

FIG. 55: Schematic of potential response elements.

FIG. 56: Genomic organization of PSM gene.

FIG. 57: Schematic of metastatic prostate cell

FIGS. 58A-58C: Nucleic acid of PSM genomic DNA is read 5 prime away fromthe transcription start site: number on the sequences indicatesnucleotide upstream from the start site. Therefore, nucleotide #121 isactually −121 using conventional numbering system (SEQ ID NO: 119).

FIG. 59: Representation of NAAG 1, acividin, azotomycin, and6-diazo-5-oxo-norleucine, DON.

FIG. 60: Preparation of N-acetylaspartylglutamate, NAAG 1.

FIG. 61:

Synthesis of N-acetylaspartylglutamate, NAAG 1.

FIG. 62: Synthesis of N-phosphonoacetylaspartyl-L-glutamate.

FIG. 63: Synthesis of 5-diethylphosphonon-2 amino benzylvalerateintermediate.

FIG. 64: Synthesis of analog 4 and 5.

FIG. 65: Representation of DON, analogs 17-20.

FIG. 66: Substrates for targeted drug delivery, analog 21 and 22.

FIG. 67: Dynemycin A and its mode of action.

FIG. 68: Synthesis of analog 28.

FIG. 69: Synthesis for intermediate analog 28.

FIG. 70: Attachment points for PALA.

FIG. 71: Mode of action for substrate 21.

FIGS. 72A-72D: Intron 1F: Forward Sequence (SEQ ID NO: 120).

FIGS. 73A-73E: Intron 1R: Reverse Sequence (SEQ ID NO: 121).

FIGS. 74A-74C: Intron 2F: Forward Sequence

FIGS. 75A-75C: Intron 2F: Forward Sequence (SEQ ID NO: 122).

FIGS. 76A-76B: Intron 3R: Forward Sequence (SEQ ID NO: 124).

FIGS. 77A-77B: Intron 3R: Reverse Sequence (SEQ ID NO: 125).

FIGS. 78A-78C: Intron 4F: Forward Sequence (SEQ ID NO: 126).

FIGS. 79A-79E: Intron 4R: Reverse Sequence (SEQ ID NO: 127)

FIG. 80: PSM genomic organization of the exons and 19 intron junctionsequences. The exon/intron junctions (See Example 15) are as follows:

-   -   1. Exon/intron 1 at bp 389-390;    -   2. Exon/intron 2 at bp 490-491;    -   3. Exon/intron 3 at bp 681-682;    -   4. Exon/intron 4 at bp 784-785;    -   5. Exon/intron 5 at bp 911-912;    -   6. Exon/intron 6 at bp 1096-1097;    -   7. Exon/intron 7 at bp 1190-1191;    -   8. Exon/intron 8 at bp 1289-1290;    -   9. Exon/intron 9 at bp 1375-1376;    -   10. Exon/intron 10 at bp 1496-1497;    -   11. Exon/intron 11 at bp 1579-1580;    -   12. Exon/intron 12 at bp 1640-1641;    -   13. Exon/intron 13 at bp 1708-1709;    -   14. Exon/intron 14 at bp 1803-1804;    -   15. Exon/intron 15 at bp 1892-1893;    -   16. Exon/intron 16 at bp 2158-2159;    -   17. Exon/intron 17 at bp 2240-2241;    -   18. Exon/intron 18 at bp 2334-2335;    -   19. Exon/intron 19 at bp 2644-2645.

SUMMARY OF THE INVENTION

This invention provides an isolated mammalian nucleic acid moleculeencoding an alternatively spliced prostate-specific membrane (PSM′)antigen.

This invention provides an isolated nucleic acid molecule encoding aprostate-specific membrane antigen promoter. This invention provides amethod of detecting hematogenous micrometastic tumor cells of a subject,and determining prostate cancer progression in a subject.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, references to specific nucleotides are tonucleotides present on the coding strand of the nucleic acid. Thefollowing standard abbreviations are used throughout the specificationto indicate specific nucleotides:

-   -   C=cytosine A=adenosine    -   T=thymidine G=guanosine

A “gene” means a nucleic acid molecule, the sequence of which includesall the information required for the normal regulated production of aparticular protein, including the structural coding sequence, promotersand enhancers.

This invention provides an isolated mammalian nucleic acid encoding analternatively spliced prostate-specific membrane (PSM′) antigen.

This invention provides an isolated mammalian nucleic acid encoding amammalian prostate-specific membrane (PSM) antigen.

This invention further provides an isolated mammalian DNA molecule of anisolated mammalian nucleic acid molecule encoding an alternativelyspliced prostate-specific membrane antigen. This invention also providesan isolated mammalian cDNA molecule encoding a mammalian alternativelyspliced prostate-specific membrane antigen. This invention provides anisolated mammalian RNA molecule encoding a mammalian alternativelyspliced prostate-specific cytosolic antigen.

This invention further provides an isolated mammalian DNA molecule of anisolated mammalian nucleic acid molecule encoding a mammalianprostate-specific membrane antigen. This invention also provides anisolated mammalian cDNA molecule encoding a mammalian prostate-specificmembrane antigen. This invention provides an isolated mammalian RNAmolecule encoding a mammalian prostate-specific membrane antigen.

In the preferred embodiment of this invention, the isolated nucleicsequence is cDNA from human as shown in FIGS. 47A-47D (SEQ ID NO: 1)This human sequence was submitted to GenBank (Los Alamos NationalLaboratory, Los Alamos, N.Mex.) with Accession Number, M99487 and thedescription as PSM, Homo sapiens, 2653 base-pairs.

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of PSM or PSM′ antigen, but whichshould not produce phenotypic changes. Alternatively, this inventionalso encompasses DNAs and cDNAs which hybridize to the DNA and cDNA ofthe subject invention. Hybridization methods are well known to those ofskill in the art.

For example, high stringent hybridization conditions are selected atabout 5° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe. Typically, stringentconditions will be those in which the salt concentration is at leastabout 0.02 molar at pH 7 and the temperature is at least about 60° C. Asother factors may significantly affect the stringency of hybridization,including, among others, base composition and size of the complementarystrands, the presence of organic solvents, ie. salt or formamideconcentration, and the extent of base mismatching, the combination ofparameters is more important than the absolute measure of any one. ForExample high stringency may be attained for example by overnighthybridization at about 68° C. in a 6×SSC solution, washing at roomtemperature with 6×SSC solution, followed by washing at about 68° C. ina 6×SSC in a 0.6×SSX solution.

Hybridization with moderate stringency may be attained for exampleby: 1) filter pre-hybridizing and hybridizing with a solution of 3×sodium chloride, sodium citrate (SSC), 50% formamide, 0.1M Tris bufferat Ph 7.5, 5× Denhardt's solution; 2.) pre-hybridization at 37° C. for 4hours; 3) hybridization at 37° C. with amount of labelled probe equal to3,000,000 cpm total for 16 hours; 4) wash in 2×SSC and 0.1% SDSsolution; 5) wash 4× for 1 minute each at room temperature at 4× at 60°C. for 30 minutes each; and 6) dry and expose to film.

The DNA molecules described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptide by a variety of recombinant techniques. The molecule isuseful for generating new cloning and expression vectors, transformedand transfected prokaryotic and eukaryotic host cells, and new anduseful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

Moreover, the isolated mammalian nucleic acid molecules encoding amammalian prostate-specific membrane antigen and the alternativelyspliced PSM′ are useful for the development of probes to study thetumorigenesis of prostate cancer.

This invention also provides an isolated nucleic acid molecule of atleast 15 nucleotides capable of specifically hybridizing with a sequenceof a nucleic acid molecule encoding the prostate-specific membraneantigen or the alternatively spliced prostate specific membrane antigen.

This nucleic acid molecule produced can either be DNA or RNA. As usedherein, the phrase “specifically hybridizing” means the ability of anucleic acid molecule to recognize a nucleic acid sequence complementaryto its own and to form double-helical segments through hydrogen bondingbetween complementary base pairs.

This nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a sequence of a nucleic acid moleculeencoding the prostate-specific membrane antigen can be used as a probe.Nucleic acid probe technology is well known to those skilled in the artwho will readily appreciate that such probes may vary greatly in lengthand may be labeled with a detectable label, such as a radioisotope orfluorescent dye, to facilitate detection of the probe. DNA probemolecules may be produced by insertion of a DNA molecule which encodesPSM antigen into suitable vectors, such as plasmids or bacteriophages,followed by transforming into suitable bacterial host cells, replicationin the transformed bacterial host cells and harvesting of the DNAprobes, using methods well known in the art. Alternatively, probes maybe generated chemically from DNA synthesizers.

RNA probes may be generated by inserting the PSM antigen moleculedownstream of a bacteriophage promoter such as T3, T7 or SP6. Largeamounts of RNA probe may be produced by incubating the labelednucleotides with the linearized PSM antigen fragment where it containsan upstream promoter in the presence of the appropriate RNA polymerase.

This invention also provides a nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of anucleic acid molecule which is complementary to the mammalian nucleicacid molecule encoding a mammalian prostate-specific membrane antigen.This molecule may either be a DNA or RNA molecule.

The current invention further provides a method of detecting theexpression of a mammalian PSM or PSM′ antigen expression in a cell whichcomprises obtaining total mRNA from the cell, contacting the mRNA soobtained with a labelled nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence of thenucleic acid molecule encoding a mammalian PSM or PSM′ antigen underhybridizing conditions, determining the presence of mRNA hybridized tothe molecule and thereby detecting the expression of the mammalianprostate-specific membrane antigen in the cell. The nucleic acidmolecules synthesized above may be used to detect expression of a PSM orPSM′ antigen by detecting the presence of mRNA coding for the PSMantigen. Total mRNA from the cell may be isolated by many procedureswell known to a person of ordinary skill in the art. The hybridizingconditions of the labelled nucleic acid molecules may be determined byroutine experimentation well known in the art. The presence of mRNAhybridized to the probe may be determined by gel electrophoresis orother methods known in the art. By measuring the amount of the hybridmade, the expression of the PSM antigen by the cell can be determined.The labeling may be radioactive. For an example, one or more radioactivenucleotides can be incorporated in the nucleic acid when it is made.

In one embodiment of this invention, nucleic acids are extracted byprecipitation from lysed cells and the mRNA is isolated from the extractusing an oligo-dT column which binds the poly-A tails of the mRNAmolecules (13). The mRNA is then exposed to radioactively labelled probeon a nitrocellulose membrane, and the probe hybridizes to and therebylabels complementary mRNA sequences. Binding may be detected byluminescence autoradiography or scintillation counting. However, othermethods for performing these steps are well known to those skilled inthe art, and the discussion above is merely an example.

This invention further provides another method to detect expression of aPSM or PSM′ antigen in tissue sections which comprises contacting thetissue sections with a labelled nucleic acid molecule of at least 15nucleotides capable of specifically hybridizing with a sequence ofnucleic acid molecules encoding a mammalian PSM antigen underhybridizing conditions, determining the presence of mRNA hybridized tothe molecule and thereby detecting the expression of the mammalian PSMor PSM′ antigen in tissue sections. The probes are also useful forin-situ hybridization or in order to locate tissues which express thisgene, or for other hybridization assays for the presence of this gene orits mRNA in various biological tissues. The in-situ hybridization usinga labelled nucleic acid molecule is well known in the art. Essentially,tissue sections are incubated with the labelled nucleid acid molecule toallow the hybridization to occur. The molecule will carry a marker forthe detection because it is “labelled”, the amount of the hybrid will bedetermined based on the detection of the amount of the marker and sowill the expression of PSM antigen.

This invention further provides isolated PSM or PSM′ antigen nucleicacid molecule operatively linked to a promoter of RNA transcription. Theisolated PSM or PSM′ antigen sequence can be linked to vector systems.Various vectors including plasmid vectors, cosmid vectors, bacteriophagevectors and other viruses are well known to ordinary skilledpractitioners. This invention further provides a vector which comprisesthe isolated nucleic acid molecule encoding for the PSM or PSM′ antigen.

As an example to obtain these vectors, insert and vector DNA can both beexposed to a restriction enzyme to create complementary ends on bothmolecules which base pair with each other and are then ligated togetherwith DNA ligase. Alternatively, linkers can be ligated to the insert DNAwhich correspond to a restriction site in the vector DNA, which is thendigested with the restriction enzyme which cuts at that site. Othermeans are also available and known to an ordinary skilled practitioner.

In an embodiment, the PSM sequence is cloned in the Not I/Sal I site ofpSPORT/vector (Gibco®-BRL). This plasmid, p55A-PSM, was deposited onAug. 14, 1992 with the American Type Culture Collection (ATCC), 12301Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganism for the Purposes of Patent Procedure. Plasmid, p55A-PSM,was accorded ATCC Accession Number 75294.

This invention further provides a host vector system for the productionof a polypeptide having the biological activity of the prostate-specificmembrane antigen. These vectors may be transformed into a suitable hostcell to form a host cell vector system for the production of apolypeptide having the biological activity of PSM antigen.

Regulatory elements required for expression include promoter sequencesto bind RNA polymerase and transcription initiation sequences forribosome binding. For example, a bacterial expression vector includes apromoter such as the lac promoter and for transcription initiation theShine-Dalgarno sequence and the start codbn AUG (14). Similarly, aeukaryotic expression vector includes a heterologous or homologouspromoter for RNA polymerase II, a downstream polyadenylation signal, thestart codon AUG, and a termination codon for detachment of the ribosome.Such vectors may be obtained commercially or assembled from thesequences described by methods well known in the art, for example themethods described above for constructing vectors in general. Expressionvectors are useful to produce cells that express the PSM antigen.

This invention further provides an isolated DNA or cDNA moleculedescribed hereinabove wherein the host cell is selected from the groupconsisting of bacterial cells (such as E.coli), yeast cells, fungalcells, insect cells and animal cells. Suitable animal cells include, butare not limited to Vero cells, HeLa cells, Cos cells, CV1 cells andvarious primary mammalian cells.

This invention further provides a method of producing a polypeptidehaving the biological activity of the prostate-specific membrane antigenwhich comprising growing host cells of a vector system containing thePSM antigen sequence under suitable conditions permitting production ofthe polypeptide and recovering the polypeptide so produced.

This invention provides a mammalian cell comprising a DNA moleculeencoding a mammalian PSM or PSM′ antigen, such as a mammalian cellcomprising a plasmid adapted for expression in a mammalian cell, whichcomprises a DNA molecule encoding a mammalian PSM antigen and theregulatory elements necessary for expression of the DNA in the mammaliancell so located relative to the DNA encoding the mammalian PSM or PSM′antigen as to permit expression thereof.

Numerous mammalian cells may be used as hosts, including, but notlimited to, the mouse fibroblast cell NIH3T3, CHO cells, HeLa cells,Ltk⁻ cells, Cos cells, etc. Expression plasmids such as that describedsupra may be used to transfect mammalian cells by methods well known inthe art such as calcium phosphate precipitation, electroporation or DNAencoding the mammalian PSM antigen may be otherwise introduced intomammalian cells, e.g., by microinjection, to obtain mammalian cellswhich comprise DNA, e.g., cDNA or a plasmid, encoding a mammalian PSMantigen.

This invention provides a method for determining whether a ligand canbind to a mammalian prostate-specific. membrane antigen which comprisescontacting a mammalian cell comprising an isolated DNA molecule encodinga mammalian prostate-specific membrane antigen with the ligand underconditions permitting binding of ligands to the mammalianprostate-specific membrane antigen, and thereby determining whether theligand binds to a mammalian prostate-specific membrane antigen.

This invention further provides ligands bound to the mammalian PSM orPSM′ antigen.

This invention also provides a therapeutic agent comprising a ligandidentified by the above-described method and a cytotoxic agentconjugated thereto. The cytotoxic agent may either be a radioisotope ora toxin. Examples of radioisotopes or toxins are well known to one ofordinary skill in the art.

This invention also provides a method of imaging prostate cancer inhuman patients which comprises administering to the patients at leastone ligand identified by the above-described method, capable of bindingto the cell surface of the prostate cancer cell and labelled with animaging agent under conditions permitting formation of a complex betweenthe ligand and the cell surface PSM or PSM′ antigen. This inventionfurther provides a composition comprising an effective imaging agent ofthe PSM OR PSM′ antigen ligand and a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are well known to one ofordinary skill in the art. For an example, such a pharmaceuticallyacceptable carrier can be physiological saline.

Also provided by this invention is a purified mammalian PSM and PSM′antigen. As used herein, the term “purified prostate-specific membraneantigen” shall mean isolated naturally-occurring prostate-specificmembrane antigen or protein (purified from nature or manufactured suchthat the primary, secondary and tertiary conformation, andposttranslational modifications are identical to naturally-occurringmaterial) as well as non-naturally occurring polypeptides having aprimary structural conformation (i.e. continuous sequence of amino acidresidues). Such polypeptides include derivatives and analogs.

This invention provides an isolated nucleic acid molecule encoding aprostate-specific membrane antigen promoter. In one embodiment the PSMpromoter has at least the sequence as in FIGS. 58A-58C.

This invention provides an isolated nucleic acid molecule encoding analternatively spliced prostate-specific membrane antigen promoter.

This invention further-provides a polypeptide encoded by the isolatedmammalian nucleic acid sequence of PSM and PSM′ antigen.

It is believed that there may be natural ligand interacting with the PSMor PSM′ antigen. This invention provides a method to identify suchnatural ligand or other ligand which can bind to the PSM or PSM′antigen. A method to identify the ligand comprises a) coupling thepurified mammalian PSM or PSM′ antigen to a solid matrix, b) incubatingthe coupled purified mammalian PSM or PSM′ protein with the potentialligands under the conditions permitting binding of ligands and thepurified PSM or PSM′ antigen; c) washing the ligand and coupled purifiedmammalian PSM or PSM′ antigen complex formed in b) to eliminate thenonspecific binding and impurities and finally d) eluting the ligandfrom the bound purified mammalian PSM or PSM′ antigen. The techniques ofcoupling proteins to a solid matrix are well known in the art. Potentialligands may either be deduced from the structure of mammalian PSM orPSM′ by other empirical experiments known by ordinary skilledpractitioners. The conditions for binding may also easily be determinedand protocols for carrying such experimentation have long been welldocumented (15).

The ligand-PSM antigen complex will be washed. Finally, the bound ligandwill be eluted and characterized. Standard ligands characterizationtechniques are well known in the art.

The above method may also be used to purify ligands from any biologicalsource. For purification of natural ligands in the cell, cell lysates,serum or other biological samples will be used to incubate with themammalian PSM or PSM′ antigen bound on a matrix. Specific natural ligandwill then be identified and purified as above described.

With the protein sequence information, antigenic areas may be identifiedand antibodies directed against these areas may be generated andtargeted to the prostate cancer for imaging the cancer or therapies.

This invention provides an antibody directed against the amino acidsequence of a mammalian PSM or PSM antigen.

The invention provides a method to select specific regions on the PSM orPSM′ antigen to generate antibodies. The protein sequence may bedetermined from the PSM or PSM′ DNA sequence. Amino acid sequences maybe analyzed by methods well known to those skilled in the art todetermine whether they produce hydrophobic or hydrophilic regions in theproteins which they build. In the case of cell membrane proteins,hydrophobic regions are well known to form the part of the protein thatis inserted into the lipid bilayer of the cell membrane, whilehydrophilic regions are located on the cell surface, in an aqueousenvironment. Usually, the hydrophilic regions will be more immunogenicthan the hydrophobic regions. Therefore the hydrophilic amino acidsequences may be selected and used to generate antibodies specific tomammalian PSM antigen. For an example, hydrophilic sequences of thehuman PSM antigen shown in hydrophilicity plot of FIGS. 16-A to 16-K maybe easily selected. The selected peptides may be prepared usingcommercially available machines. As an alternative, DNA, such as a cDNAor a fragment thereof, may be cloned and expressed and the resultingpolypeptide recovered and used as an immunogen.

Polyclonal antibodies against these peptides may be produced byimmunizing animals using the selected peptides. Monoclonal antibodiesare prepared using hybridoma technology by fusing antibody producing Bcells from immunized animals with myeloma cells and selecting theresulting hybridoma cell line producing the desired antibody.Alternatively, monoclonal antibodies may be produced by in vitrotechniques known to a person of ordinary skill in the art. Theseantibodies are useful to detect the expression of mammalian PSM antigenin living animals, in humans, or in biological tissues or fluidsisolated from animals or humans.

In one embodiment, peptides Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID No.34),Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No.35) and Lys-Ser-Pro-Asp-Glu-Gly (SEQID No36) of human PSM antigen are selected.

This invention further provides polyclonal and monoclonal antibody(ies)against peptides Asp-Glu-Leu-Lys-Ala-Glu (SEQ ID No. 34)Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID No. 35) and Lys-Ser-Pro-Asp-Glu-Gly (SEQID No. 36).

This invention provides a therapeutic agent comprising antibodies orligand(s) directed against PSM antigen and a cytotoxic agent conjugatedthereto or antibodies linked enzymes which activate prodrug to kill thetumor. The cytotoxic agent may either be a radioisotope or toxin.

This invention provides a method of imaging prostate cancer in humanpatients which comprises administering to the patient the monoclonalantibody directed against the peptide of the mammalian PSM or PSM′antigen capable of binding to the cell surface of the prostate cancercell and labeled with an imaging agent under conditions permittingformation of a complex between the monoclonal antibody and the cellsurface prostate-specific membrane antigen. The imaging agent is aradioisotope such as Indium¹¹¹.

This invention further provides a prostate cancer specific imaging agentcomprising the antibody directed against PSM or PSM′ antigen and aradioisotope conjugated thereto.

This invention also provides a composition comprising an effectiveimaging amount of the antibody directed against the PSM or PSM′ antigenand a pharmaceutically acceptable carrier. The methods to determineeffective imaging amounts are well known to a skilled practitioner. Onemethod is by titration using different amounts of the antibody.

This invention further provides an immunoassay for measuring the amountof the prostate-specific membrane antigen in a biological samplecomprising steps of a) contacting the biological sample with at leastone antibody directed against the PSM or PSM′ antigen to form a complexwith said antibody and the prostate-specific membrane antigen, and b)measuring the amount of the prostate-specific membrane antigen in saidbiological sample by measuring the amount of said complex. One exampleof the biological sample is a serum sample.

This invention provides a method to purify mammalian prostate-specificmembrane antigen comprising steps of a) coupling the antibody directedagainst the PSM or PSM′ antigen to a solid matrix; b) incubating thecoupled antibody of a) with lysate containing prostate-specific membraneantigen under the condition which the antibody and prostate membranespecific can bind; c) washing the solid matrix to eliminate impuritiesand d) eluting the prostate-specific membrane antigen from the coupledantibody.

This invention also provides a transgenic nonhuman mammal whichcomprises the isolated nucleic acid molecule encoding a mammalian PSM orPSM′ antigen. This invention further provides a transgenic nonhuman.mammal whose genome comprises antisense DNA complementary to DNAencoding a mammalian prostate-specific membrane antigen so placed as tobe transcribed into antisense mRNA complementary to mRNA encoding theprostate-specific membrane antigen and which hybridizes to mRNA encodingthe prostate specific antigen thereby reducing its translation.

Animal model systems which elucidate the physiological and behavioralroles of mammalian PSM or PSM′ antigen are produced by creatingtransgenic animals in which the expression of the PSM or PSM′ antigen iseither increased or decreased, or the amino acid sequence of theexpressed PSM antigen is altered, by a variety of techniques. Examplesof these techniques include, but are not limited to: 1) Insertion ofnormal or mutant versions of DNA encoding a mammalian PSM or PSM′antigen, by microinjection, electroporation, retroviral transfection orother means well known to those skilled in the art, into appropriatefertilized embryos in order to produce a transgenic animal (16) or 2)Homologous recombination (17) of mutant or normal, human or animalversions of these genes with the native gene locus in transgenic animalsto alter the regulation of expression or the structure of these PSM orPSM′ antigen sequences. The technique of homologous recombination iswell known in the art. It replaces the native gene with the insertedgene and so is useful for producing an animal that cannot express nativePSM antigen but does express, for example, an inserted mutant PSMantigen, which has replaced the native PSM antigen in the animal'sgenome by recombination, resulting in undere xpression of thetransporter. Microinjection adds genes to the genome, but does notremove them, and so is useful for producing an animal which expressesits own and added PSM antigens, resulting in over expression of the PSMantigens.

One means available for producing a transgenic animal, with a mouse asan example, is as follows: Female mice are mated, and the resultingfertilized eggs are dissected out of their oviducts. The eggs are storedin an appropriate medium such as Me medium (16). DNA or cDNA encoding amammalian PSM antigen is purified from a vector by methods well known inthe art. Inducible promoters may be fused with the coding region of theDNA to provide an experimental means to regulate expression of thetrans-gene. Alternatively or in addition, tissue specific regulatoryelements may be fused with the coding region to permit tissue-specificexpression of the trans-gene. The DNA, in an appropriately bufferedsolution, is put into a microinjection needle (which may be made fromcapillary tubing using a pipet puller) and the egg to be injected is putin a depression slide. The needle is inserted into the pronucleus of theegg, and the DNA solution is injected. The injected egg is thentransferred into the oviduct of a pseudopregnant mouse (a mousestimulated by the appropriate hormones to maintain pregnancy but whichis not actually pregnant), where it proceeds to the uterus, implants,and develops to term. As noted above, microinjection is not the onlymethod for inserting DNA into the egg cell, and is used here only forexemplary purposes.

Another use of the PSM antigen sequence is to isolate homologous gene orgenes in different mammals. The gene or genes can be isolated by lowstringency screening of either cDNA or genomic libraries of differentmammals using probes from PSM sequence. The positive clones identifiedwill be further analyzed by DNA sequencing techniques which are wellknown to an ordinary person skilled in the art. For example, thedetection of members of the protein serine kinase family by homologyprobing.

This invention provides a method of suppressing or modulating metastaticability of prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells comprising introducing a DNA molecule encoding aprostate specific membrane antigen operatively linked to a 5′ regulatoryelement into a tumor cell of a subject, in a way that expression of theprostate specific membrane antigen is under the control of theregulatory element, thereby suppressing or modulating metastatic abilityof prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells. The subject may be a mammal or more specifically ahuman.

In one embodiment, the DNA molecule encoding prostate specific membraneantigen operatively linked to a 5′ regulatory element forms part of atransfer vector which is inserted into a cell or organism. In additionthe vector is capable or replication and expression of prostate specificmembrane antigen. The DNA molecule encoding prostate specific membraneantigen can be integrated into a genome of a eukaryotic or prokaryoticcell or in a host cell containing and/or expressing a prostate specificmembrane antigen.

Further, the DNA molecule encoding prostate specific membrane antigenmay be introduced by a bacterial, viral, fungal, animal, or liposomaldelivery vehicle. Other means are also available and known to anordinary skilled practitioner.

Further, the DNA molecule encoding a prostate specific membrane antigenoperatively linked to a promoter or enhancer. A number of viral vectorshave been described including those made from various promoters andother regulatory elements derived from virus sources. Promoters consistof short arrays of nucleic acid sequences that interact specificallywith cellular proteins involved in transcription. The combination ofdifferent recognition sequences and the cellular concentration of thecognate transcription factors determines the efficiency with which agene is transcribed in a particular cell type.

Examples of suitable promoters include a viral promoter. Viral promotersinclude: adenovirus promoter, an simian virus 40 (SV40) promoter, acytomegalovirus (CMV) promoter, a mouse mammary tumor virus (MMTV)promoter, a Malony murine leukemia virus promoter, a murine sarcomavirus promoter, and a Rous sarcoma virus promoter.

Further, another suitable promoter is a heat shock promoter.Additionally, a suitable promoter is a bacteriophage promoter. Examplesof suitable bacteriophage promoters include but not limited to, a T7promoter, a T3 promoter, an SP6 promoter, a lambda promoter, abaculovirus promoter.

Also suitable as a promoter is an animal cell promoter such as aninterferon promoter, a metallothionein promoter, an immunoglobulinpromoter. A fungal promoter is also a suitable promoter. Examples offungal promoters include but are not limited to, an ADC1 promoter, anARG promoter, an ADH promoter, a CYC1 promoter, a CUP promoter, an ENO1promoter, a GAL promoter, a PHO promoter, a PGK promoter, a GAPDHpromoter, a mating type factor promoter. Further, plant cell promotersand insect cell promoters are also suitable for the methods describedherein.

This invention provides a method of suppressing or modulating metastaticability of prostate tumor cells, prostate tumor growth or elimination ofprostate tumor cells, comprising introducing a DNA molecule encoding aprostate specific membrane antigen operatively linked to a 5′ regulatoryelement coupled with a therapeutic DNA into a tumor cell of a subject,thereby suppressing or modulating metastatic ability of prostate tumorcells, prostate tumor growth or elimination of prostate tumor cells. Thesubject may be a mammal or more specifically a human.

Further, the therapeutic DNA which is coupled to the DNA moleculeencoding a prostate specific membrane antigen operatively linked to a 5′regulatory element into a tumor cell may code for a cytokine, viralantigen, or a pro-drug activating enzyme. Other means are also availableand known to an ordinary skilled practitioner.

In addition, this invention provides a prostate tumor cell, comprising aDNA molecule isolated from mammalian nucleic acid encoding a mammalianprostate-specific membrane antigen under the control of a prostatespecific membrane antigen operatively linked to a 5′ regulatory element.

As used herein, DNA molecules include complementary DNA (cDNA),synthetic DNA, and genomic DNA.

This invention provides a therapeutic vaccine for preventing humanprostate tumor growth or stimulation of prostate tumor cells in asubject, comprising administering an effective amount to the prostatecell, and a pharmaceutical acceptable carrier, thereby preventing thetumor growth or stimulation of tumor cells in the subject. Other meansare also available and known to an ordinary skilled practitioner.

This invention provides a method of detecting hematogenous micrometastictumor cells of a subject, comprising (A) performing nested polymerasechain reaction (PCR) on blood, bone marrow or lymph node samples of thesubject using the prostate specific membrane antigen primers oralternatively spliced prostate specific antigen primers, and (B)verifying micrometastases by DNA sequencing and Southern analysis,thereby detecting hematogenous micrometastic tumor cells of the subject.The subject may be a mammal or more specifically a human.

The micrometastatic tumor cell may be a prostatic cancer and the DNAprimers may be derived from prostate specific antigen. Further, thesubject may be administered with simultaneously an effective amount ofhormones, so as to increase expression of prostate specific membraneantigen. Further, growth factors or cytokine may be administered inseparately or in conjunction with hormones. Cytokines include, but arenot limited to: transforming growth factor beta, epidermal growth factor(EGF) family, fibroblast growth factors, hepatocyte growth factor,insulin-like growth factors, B-nerve growth factor, platelet-derivedgrowth factor, vascular endothelial growth factor, interleukin 1, IL-1receptor antagonist, interleukin 2, interleukin 3, interleukin 4,interleukin 5, interleukin 6, IL-6 soluble receptor, interleukin 7,interleukin 8, interleukin 9, interleukin 10, interleukin 11,interleukin 12, interleukin 13, angiogenin, chemokines, colonystimulating factors, granulocyte-macrophage colony stimulating factors,erythropoietin, interferon, interferon gamma, leukemia inhibitoryfactor, oncostatin M, pleiotrophin, secretory leukocyte proteaseinhibitor, stem cell factor, tumor necrosis factors, adhesion molecule,and soluble tumor necrosis factor (TNF) receptors.

This invention provides a method of abrogating the mitogenic responsedue to transferrin, comprising introducing a DNA molecule encodingprostate specific membrane antigen operatively linked to a 5′ regulatoryelement into a tumor cell, the expression of which gene is directlyassociated with a defined pathological effect within a multicellularorganism, thereby abrogating mitogen response due to transferrin. Thetumor cell may be a prostate cell.

This invention provides a method of determining prostate cancerprogression in a subject which comprises: a) obtaining a suitableprostate tissue sample; b) extracting RNA from the prostate tissuesample; c) performing a RNAse protection assay on the RNA therebyforming a duplex RNA-RNA hybrid; d) detecting PSM and PSM′ amounts inthe tissue sample; e) calculating a PSM/PSM′ tumor index, therebydetermining prostate cancer progression in the subject. In-situhyribridization may be performed in conjunction with the above detectionmethod.

This invention provides a method of detecting prostate cancer in asubject which comprises: (a) obtaining from a subject a prostate tissuesample; (b) treating the tissue sample so as to separately recovernucleic acid molecules present in the prostate tissue sample; (c)contacting the resulting nucleic acid molecules with multiple pairs ofsingle-stranded labeled oligonucleotide primers, each such pair beingcapable of specifically hybridizing to the tissue sample, underhybridizing conditions; (d) amplifying any nucleic acid molecules towhich a pair of primers hybridizes so as to obtain a double-strandedamplification product; (e) treating any such double-strandedamplification product so as to obtain single-stranded nucleic acidmolecules therefrom; (f) contacting any resulting single-strandednucleic acid molecules with multiple single-stranded labeledoligonucleotide probes, each such probe containing the same label andbeing capable of specifically hybridizing with such tissue sample, underhybridizing conditions; (g) contacting any resulting hybrids with anantibody to which a marker is attached and which is capable ofspecifically forming a complex with the labeled-probe, when the probe ispresent in such a complex, under complexing conditions; and (h)detecting the presence of any resulting complexes, the presence thereofbeing indicative of prostate cancer in a subject.

This invention provides a method of enhancing antibody based targetingof PSM or PSM′ in prostate tissue for diagnosis or therapy of prostatecancer comprising administering to a patient b-FGF in sufficient amountto cause upregulation of PSM or PSM′ expression.

This invention provides a method of enhancing antibody based targetingof PSM or PSM′ in prostate tissue for diagnosis or therapy of prostatecancer comprising administering to a patient TGF in sufficient amount tocause upregulation of PSM expression or PSM′.

This invention provides a method of enhancing antibody based targetingof PSM or PSM′ in prostate tissue for diagnosis or therapy of prostatecancer comprising administering to a patient EGF in sufficient amount tocause upregulation of PSM or PSM′ expression.

This invention provides a pharmaceutical composition comprising aneffective amount of PSM or the alternatively spliced PSM and a carrieror diluent. Further, this invention provides a method for administeringto a subject, preferably a human, the pharmaceutical composition.Further, this invention provides a composition comprising an amount ofPSM or the alternatively spliced PSM and a carrier or diluent.Specifically, this invention may be used as a food additive.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. Precise amountsof active ingredient required to be administered depend on the judgmentof the practitioner and are peculiar to each subject.

Suitable regimes for initial administration and booster shots are alsovariable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration.

As used herein administration means a method of administering to asubject. Such methods are well known to those skilled in the art andinclude, but are not limited to, administration topically, parenterally,orally, intravenously, intramuscularly, subcutaneously or by aerosol.Administration of PSM may be effected continuously or intermittently.

The pharmaceutical formulations or compositions of this invention may bein the dosage form of solid, semi-solid, or liquid such as, e.g.,suspensions, aerosols or the like. Preferably the compositions areadministered in unit dosage forms suitable for single administration ofprecise dosage amounts. The compositions may also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological saline, Ringer's solution, dextrose solution, and Hank'ssolution. In addition, the pharmaceutical composition or formulation mayalso include other carriers, adjuvants; or nontoxic, nontherapeutic,nonimmunogenic stabilizers and the like. Effective amounts of suchdiluent or carrier are those amounts which are effective to obtain apharmaceutically acceptable formulation in terms of solubility ofcomponents, or biological activity, etc

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claim which followthereafter.

EXPERIMENTAL DETAILS Example 1

Materials and Methods:

The approach for cloning the gene involved purification of the antigenby immunoprecipitation, and microsequencing of several internal peptidesfor use in synthesizing degenerate oligonucleotide primers forsubsequent use in the polymerase chain reaction (19, 20). A partial cDNAwas amplified as a PCR product and this was used as a homologous probeto clone the full-length cDNA molecule from a LNCaP (Lymph NodeCarcinoma of Prostate) cell line cDNA plasmid library (8).

Western Analysis of the PSH Antigen:

Membrane proteins were isolated from cells by hypotonic lysis followedby centrifugation over a sucrose density gradient (21). 10-20 μg ofLNCaP, DU-145, and PC-3 membrane proteins were electrophoresed through a10% SDS-PAGE resolving gel with a 4% stacking gel at 9-10 milliamps for16-18 hours. Proteins were electroblotted onto PVDF membranes(Millipore® Corp.) in transfer buffer (48 mM Tris base, 39 mM Glycine,20% Methanol) at 25 volts overnight at 4° C. Membranes were blocked inTSB (0.15M NaCl, 0.01M Tris base, 5% BSA) for 30 minutes at roomtemperature followed by incubation with 10-15 μg/ml of CYT-356monoclonal antibody (cytogen Corp.) for 2 hours. Membranes were thenincubated with 10-15 μg/ml of rabbit anti-mouse immunoglobulin (AccurateScientific) for 1 hour at room temperature followed by incubation with¹²⁵I-Protein A (Amersham®) at 1×10⁶ cpm/ml at room temperature.Membranes were then washed and autoradiographed for 12-24 hours at −70°C. (FIG. 1).

Immunohistochemical Analysis of PSM Antigen Expression:

The avidin-biotin method of immunohistochemical detection was employedto analyze both human tissue sections and cell lines for PSM Antigenexpression (22). Cryostat-cut prostate tissue sections (4-6 μm thick)were fixed in methanol/acetone for 10 minutes. Cell cytospins were madeon glass slides using 50,000 cells/100 μl/slide. Samples were treatedwith 1% hydrogen peroxide in PBS for 10-15 minutes in order to removeany endogenous peroxidase activity. Tissue sections were washed severaltimes in PBS, and then incubated with the appropriate suppressor serumfor 20 minutes. The suppressor serum was drained off and the sections orcells were then incubated with the diluted CYT-356 monoclonal antibodyfor 1 hour. Samples were then washed with PBS and sequentially incubatedwith secondary antibodies (horse or goat immunoglobulins, 1:200 dilutionfor 30 minutes), and with avidin-biotin complexes (1:25 dilution for 30minutes). DAB was used as a chromogen, followed by hematoxylincounterstaining and mounting. Frozen sections of prostate samples andduplicate cell cytospins were used as controls for each experiment. As apositive control, the anti-cytokeratin monoclonal antibody CAM 5.2 wasused following the same procedure described above. Tissue sections areconsidered by us to express the PSM antigen if at least 5% of the cellsdemonstrate immunoreactivity. The scoring system is as follows: 1=<5%;2=5-19%; 3=20-75%; and 4=>75% positive cells. Homogeneity versusheterogeneity was accounted for by evaluating positive and negativecells in 3-5 high power light microscopic fields (400×), recording thepercentage of positive cells among 100-500 cells. The intensity ofimmunostaining is graded on a 1+ to 4+ scale, where 1+ represents mild,2-3+ represents moderate, and 4+ represents intense immunostaining ascompared to positive controls.

Immunoprecipitation of the PSM Antigen:

80%-confluent LNCaP cells in 100 mm petri dishes were starved in RPMImedia without methionine for 2 hours, after which ³⁵S-Methionine wasadded at 100 μCi/ml and the cells were grown for another 16-18 hours.Cells were then washed and lysed by the addition of 1 ml of lysis buffer(1% Triton X-100, 50 mM Hepes pH 7.5, 10% glycerol, 150 mM MgCl₂, 1 mMPMSF, and 1 mM EGTA) with incubation for 20 minutes at 4° C. Lysateswere pre-cleared by mixing with Pansorbin® cells (Calbiochem®) for 90minutes at 4° C. Cell lysates were then mixed with Protein A Sepharose®CL-4B beads (Pharmacia®) previously bound with CYT-356 antibody (CytogenCorp.) and RAM antibody (Accurate Scientific) for 3-4 hours at 4° C. 12μg of antibody was used per 3 mg of beads per petri dish. Beads werethen washed with HNTG buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 0.1%Triton X-100, 10% glycerol, and 2 mM Sodium Orthovanadate), resuspendedin sample loading buffer containing β-mercaptoethanol, denatured at 95°C. for 5-10 minutes and run on a 10% SDS-PAGE gel with a 4° stacking gelat 10 milliamps overnight. Gels were stained with Coomassie Blue,destained with acetic acid/methanol, and dried down in a vacuum dryer at60° C. Gels were then autoradiographed for 16-24 hours at −70° C. (FIGS.2A-2D).

Immunoprecipitation and Peptide Sequencing:

The procedure described above for immunoprecipitation was repeated with8 confluent petri dishes containing approximately 6×10⁷ LNCaP cells. Theimmunoprecipitation product was pooled and loaded into two lanes of a10% SDS-PAGE gel and electrophoresed at 9-10 milliamps for 16 hours.Proteins were electroblotted onto Nitrocellulose BA-85 membranes(Schleicher and Schuell®) for 2 hours at 75 volts at 4° C. in transferbuffer. Membranes were stained with Ponceau Red to visualize theproteins and the 100 kD protein band was excised, solubilized, anddigested proteolytically with trypsin. HPLC was then performed on thedigested sample on an Applied Biosystems Model 171C and clear dominantpeptide peaks were selected and sequenced by modified Edman degradationon a modified post liquid Applied Biosystems Model 477A Protein/PeptideMicrosequencer (23). Sequencing data on all of the peptides is includedwithin this document. The amino-terminus of the PSM antigen wassequenced by a similar method which involved purifying the antigen byimmunoprecipitation and transfer via electro-blotting to a PVDF membrane(Millipore®). Protein was analyzed on an Applied Biosystems Model 477AProtein/Peptide Sequencer and the amino terminus was found to beblocked, and therefore no sequence data could be obtained by thistechnique.

PSMA Antigen Peptide Sequences:

-   2T17 #5 SLYES (W) TK (SEQ ID. NO: 2).-   2T22 #9 (S) YPDGXNLPGG (g) VQR (SEQ ID NO: 3).-   2T26 #3 FYDPMFK (SEQ ID NO: 4).-   2T27 #4 IYNVIGTL (K) (SEQ ID NO: 5).-   2T34 #6 FLYXXTQIPHLAGTEQNFQLAK (SEQ ID NO:6).-   2T35 #2 G/PVILYSDPADYFAPD/GVK (SEQ ID NO: 7-8).-   2T38 #1 AFIDPLGLPDRPFYR (SEQ ID NO: 9).-   2T46 #8 YAGESFPGIYDALFDIESK (SEQ ID NO: 10).-   2T47 #7 TILFAS(W)DAEEFGXX(Q)STE(E)A(e) (SEQ ID NO:11).

Notes: X means that no residue could be identified at this position.Capital denotes identification but with a lower degree of confidence.(lower case) means residue present but at very low levels. . . .indicates sequence continues but has dropped below detection limit.

All of these peptide sequences were verified to be unique after acomplete homology search of the translated Genbank computer database.

Degenerate PCR:

Sense and anti-sense 5′-unphosphorylated degenerate oligonucleotideprimers 17 to 20 nucleotides in length corresponding to portions of theabove peptides were synthesized on an Applied Biosystems Model 394A DNASynthesizer. These primers have degeneracies from 32 to 144. The primersused are shown below. The underlined amino acids in the peptidesrepresent the residues used in primer design.

Peptide 3: FYDPMFK (SEQ ID No. )

PSM Primer “A” TT (C or T)-TA (C or T)-GA (C or T)-CCX-ATG-TT (SEQ IDNO: 12)

PSM Primer “B” AAC-ATX-GG (A or G)-TC (A or G)-TA (A or G)-AA (SEQ IDNO:13).

Primer A is sense primer and B is anti-sense. Degeneracy is 32-fold.

Peptide 4: IYNVIGTL (K) (SEQ ID NO: 5).

PSM Primer “C” AT (T or C or A)-TA (T or C)-AA (T or C)-GTM-AT (T or Cor A)-GG (SEQ ID NO: 14).

PSM Primer “D” CC (A or T or G)-ATX-AC (G or A)-TT (A or G)-TA (A or Gor T)-AT (SEQ ID NO:15)

Primer C is sense primer and D is anti-sense. Degeneracy is 144-fold.

Peptide 2: G/PVILYSDPADYFAPD/GVK (SEQ ID NO:7-8)

PSM Primer “E” CCX-GCX-GA (T or C)-TA (T or C)-TT (T or C)-GC (SEQ IDNO: 16).

PSM Primer “F” GC (G or A)-AA (A or G)-TA (A or G)-TXC-GCX-GG (SEQ IDNO:17).

Primer E is sense primer and F is antisense primer. Degeneracy is128-fold.

Peptide 6: FLYXXTQIPHLAGTEONFOLAK (SEQ ID No. )

PSM Primer “I” ACX-GA (A or G)-CA (A or G)-AA (T or C)-TT (T or C)-CA (Aor G)-CT (SEQ ID NO: 18).

PSM Primer “J” AG-(T or C) TG-(A or G) AA-(A or G) TT-(T or C)-TG (T orC)-TC-XGT (SEQ ID NO: 19).

PSM Primer “K” GA (A or G)-CA (A or G)-AA (T or C)-TT (T or C) CA (A orG)-CT (SEQ ID NO: 20).

PSM Primer “L” AG-(T or C) TG-(A or G) AA-(A or G) TT-(T or C) TG-(T orC) TC (SEQ ID NO: 21).

Primers I and K are sense primers and J and L are anti-sense. I and Jhave degeneracies of 128-fold and K and L have 32-fold degeneracy.

Peptide 7:TILFAS (W)DAEEFGXX (q)STE (e) A (E) . . . (SEQ ID NO: 11)

PSM Primer “M” TGG-GA (T or C)-GCX-GA (A or G)-GA (A or G)-TT (C orT)-GG (SEQ ID NO: 22).

PSM Primer “N” CC-(G or A) AA-(T or C) TC-(T or C) TC-XGC-(A or G)TC-CCA (SEQ ID NO: 23).

PSM Primer “O” TGG-GA(T or C)-GCX-GA(A or G)-GA (A or G)-TT (SEQ ID NO:24).

PSM Primer “P” AA-(T or C) TC-(T or C) TC-XGC-(A or G) TC-CCA (SEQ IDNO: 25).

Primers M and O are sense primers and N and P are anti-sense. M and Nhave degeneracy of 64-fold and O and P are 32-fold degenerate.

Degenerate PCR was performed using a Perkin-Elmer Model 480 DNA thermalcycler. cDNA template for the PCR was prepared from LNCaP mRNA which hadbeen isolated by standard methods of oligo dT chromatography(Collaborative Research). The cDNA synthesis was carried out as follows:

4.5 μl LNCaP poly A+ RNA (2 μg) 1.0 μl Oligo dT primers (0.5 μg) 4.5 μldH₂O 10 μl

Incubate at 68° C.×10 minutes.

Quick chill on ice×5 minutes.

Add:

4 μ1 5 × RT Buffer 2 μ l 0.1 M DTT 1 μ l 10 mM dNTPs 0.5 μ l RNasin(Promega) 1.5 μl dH₂O 19 μ l

Incubate for 2 minutes at 37° C.

Add 1 μl Superscript® Reverse Transcriptase (Gibco®-BRL) Incubate for 1hour at 37° C.

Add 30 μl dH₂O.

Use 2 μl per PCR reaction.

Degenerate PCR reactions were optimized by varying the annealingtemperatures, Mg++ concentrations, primer concentrations, buffercomposition, extension times and number of cycles. The optimal thermalcycler profile was: Denaturation at 94° C.×30 seconds, Annealing at45-55° C. for 1 minute (depending on the mean T_(m) of the primersused), and Extension at 72° C. for 2 minutes.

5 μl 10 × PCR Buffer* 5 μl 2.5 mM dNTP Mix 5 μl Primer Mix (containing0.5-1.0 μg each of sense and anti-sense primers) 5 μl 100 mMβ-mercaptoethanol 2 μl LNCaP cDNA template 5 μl 25 mM MgCl₂ (2.5 mMfinal) 21 μl dH₂O 2 μl diluted Taq Polymerase (0.5 U/μl) 50 μl totalvolume

Tubes were overlaid with 60 μl of light mineral oil and amplified for 30cycles. PCR products were analyzed by electrophoresing 5 μl of eachsample on a 2-3% agarose gel followed by staining with Ethidium bromideand photography.

*10 × PCR Buffer 166 mM NH₄SO₄ 670 mM Tris, pH 8.8 2 mg/ml BSA

Representative photographs displaying PCR products are shown in FIG. 5.

Cloning of PCR Products:

In order to further analyze these PCR products, these products werecloned into a suitable plasmid vector using “TA Cloning” (Invitrogen®Corp.). The cloning strategy employed here is to directly ligate PCRproducts into a plasmid vector possessing overhanging T residues at theinsertion site, exploiting the fact that Taq polymerase leavesoverhanging A residues at the ends of the PCR products. The ligationmixes are transformed into competent E. coli cells and resultingcolonies are grown up, plasmid DNA is isolated by the alkaline lysismethod (24), and screened by restriction analysis (FIGS. 6A-6B).

DNA Sequencing of PCR Products:

TA Clones of PCR products were then sequenced by the dideoxy method (25)using Sequenase (U.S. Biochemical). 3-4 μg of each plasmid DNA wasdenatured with NaOH and ethanol precipitated. Labeling reactions werecarried out as per the manufacturers recommendations using ³⁵S-ATP, andthe reactions were terminated as per the same protocol. Sequencingproducts were then analyzed on 6% polyacrylamide/7M Urea gels using anIBI sequencing apparatus. Gels were run at 120 watts for 2 hours.Following electrophoresis, the gels were fixed for 15-20 minutes in 10%methanol/10% acetic acid, transferred onto Whatman 3MM paper and drieddown in a Biorad® vacuum dryer at 80° C. for 2 hours. Gels were thenautoradiographed at room temperature for 16-24 hours. In order todetermine whether the PCR products were the correct clones, thesequences obtained at the 5′ and 3′ ends of the molecules were analyzedfor the correct primer sequences, as well as adjacent sequences whichcorresponded to portions of the peptides not used in the design of theprimers.

IN-20 was confirmed to be correct and represent a partial cDNA for thePSM gene. In this PCR reaction, I and N primers were used. The DNAsequence reading from the I primer was:

ACG GAG CAA AAC TTT CAG CTT GCA AAG (SEQ ID NO:29).

T E O N F O LAK (SEQ ID NO: 30)

The underlined amino acids were the portion of peptide 6 that was usedto design this sense primer and the remaining amino acids which agreewith those present within the peptide confirm that this end of themolecule represents the correct protein (PSM antigen).

When analyzed the other end of the molecule by reading from the N primerthe anti-sense sequence was:

CTC TTC GGC ATC CCA GCT TGC AAA CAA AAT TGT TCT (SEQ ID NO: 31)

Sense (complementary) Sequence:

AGA ACA ATT TTG TTT GCA AGC TGG GAT GCC AAG GAG (SEQ ID NO: 32)

R T I L F A S W D A EE (SEQ ID NO: 33)

The underlined amino acids here represent the portion of peptide 7 usedto create primer N. All of the amino acids upstream of this primer arecorrect in the IN-20 clone, agreeing with the amino acids found inpeptide 7. Further DNA sequencing has enabled us to identify thepresence of other PSM peptides within the DNA sequence of the positiveclone.

The DNA sequence of this partial cDNA was found to be unique whenscreened on the Genbank computer database.

cDNA Library Construction and Cloning of Full—Length PSM cDNA:

A cDNA library from LNCaP mRNA was constructed using the Superscriptsplasmid system (BRL®-Gibco). The library was transformed using competentDH5-α cells and plated onto 100 mm plates containing LB plus 100 μg/mlof Carbenicillin. Plates were grown overnight at 37° C. and colonieswere transferred to nitrocellulose filters. Filters were processed andscreened as per Grunstein and Hogness (26), using the 1.1 kb partialcDNA homologous probe which was radiolabelled with ³²P-dCTP by randompriming (27). Eight positive colonies were obtained which upon DNArestriction and sequencing analysis proved to represent full-length cDNAmolecules coding for the PSM antigen. Shown in FIG. 7 is anautoradiogram showing the size of the cDNA molecules represented in thelibrary and in FIG. 8 restriction analysis of several full-length clonesis shown. FIG. 9 is a plasmid Southern analysis of the samples in FIG.8, showing that they all hybridize to the 1.1 kb partial cDNA probe.

Both the cDNA as well as the antigen have been screened through theGenbank Computer database (Human Genome Project) and have been found tobe unique.

Northern Analysis of PSM Gene Expression:

Northern analysis (28) of the PSM gene has revealed that expression islimited to the prostate and to prostate carcinoma.

RNA samples (either 10 μg of total RNA or 2 μg of poly A+ RNA) weredenatured and electrophoresed through 1.1% agarose/formaldehyde gels at60 milliamps for 6-8 hours. RNA was then transferred to Nytran® nylonmembranes (Schleicher and Schuell®) by pressure blotting in 10×SSC witha Posi-blotter (Stratagene®). RNA was cross-linked to the membranesusing a Stratalinker (Stratagene®) and subsequently baked in a vacuumoven at 80° C. for 2 hours. Blots were pre-hybridized at 65° C. for 2hours in prehybridization solution (BRL®) and subsequently hybridizedfor 16 hours in hybridization buffer (BRL®) containing 1-2×10⁶ cpm/ml of³² P-labelled random-primed cDNA probe. Membranes were washed twice in1×SSPE/1% SDS and twice in 0.1×SSPE/1% SDS at 42° C. Membranes were thenair-dried and autoradiographed for 12-36 hours at −70° C.

PCR Analysis of PSM Gene Expression in Human Prostate Tissues:

PCR was performed on 15 human prostate samples to determine PSM geneexpression. Five samples each from normal prostate tissue, benignprostatic hyperplasia, and prostate cancer were used (histologyconfirmed by MSKCC Pathology Department).

10 μg of total RNA from each sample was reverse transcribed to made cDNAtemplate as previously described in section IV. The primers usedcorresponded to the 5′ and 3′ ends of the 1.1 kb partial cDNA, IN-20,and therefore the expected size of the amplified band is 1.1 kb. Sincethe T_(m) of the primers is 64° C. PCR primers were annealed at 60° C.PCR was carried out for 35 cycles using the same conditions previouslydescribed in section IV.

LNCaP and H26-Ras transfected LNCaP (29) were included as a positivecontrol and DU-145 as a negative control. 14/15 samples clearlyamplified the 1.1 kb band and therefore express the gene.

Experimental Results

The gene which encodes the 100 kD PSM antigen has been identified. Thecomplete cDNA is shown in Sequence ID #1. Underneath that nucleic acidsequence is the predicted translated amino acid sequence. The totalnumber of the amino acids is 750, ID #2.

The hydrophilicity of the predicted protein sequence is shown in FIGS.16-A to 16-K. Shown in FIGS. 17A-17C are three peptides with the highestpoint of hydrophilicity. They are: Asp-Glu-Leu-Lys-Ala-Glu (SEQ IDNO:34); Asn-Glu-Asp-Gly-Asn-Glu (SEQ ID NO:35); andLys-Ser-Pro-Asp-Glu-Gly (SEQ ID NO: 36)

By the method of Klein, Kanehisa and DeLisi, a specificmembrane—spanning domain is identified. The sequence is from the aminoacid #19 to amino acid #44:Ala-Gly-Ala-Leu-Val-Leu-Ala-Gly-Gly-Phe-Phe-Leu-Leu-Gly-Phe-Leu-Phe (SEQID NO: 37)

This predicted membrane-spanning domain was computed on PC Gene(computer software program). This data enables prediction of inner andouter membrane domains of the PSM antigen which aids in designingantibodies for uses in targeting and imaging prostate cancer.

When the PSM antigen sequence with other known sequences of the GeneBankwere compared, homology between the PSM antigen sequence and thetransferrin receptor sequence were found. The data are shown in FIG. 18.

Experimental Discussions

Potential Uses for PSM Antigen:

1. Tumor Detection:

Microscopic:

Unambiguous tumor designation can be accomplished by use of probes fordifferent antigens. For prostatic cancer, the PSM antigen probe mayprove beneficial. Thus PSM could be used for diagnostic purposes andthis could be accomplished at the microscopic level using in-situhybridization using sense (control) and antisense probes derived fromthe coding region of the cDNA cloned by the applicants. This could beused in assessment of local extraprostatic extension, involvement oflymph node, bone or other metastatic sites. As bone metastasis presentsa major problem in prostatic cancer, early detection of metastaticspread is required especially for staging. In some tumors detection oftumor cells in bone marrow portendsa grim prognosis and suggests thatinterventions aimed at metastasis be tried. Detection of PSM antigenexpression in bone marrow aspirates or sections may provide such earlyinformation. PCR amplification or in-situ hybridization may be used.Using RT-PCR cells in the circulating can be detected by hematogenousmetastasis.

2. Antigenic Site Identification

The knowledge of the cDNA for the antigen also provides for theidentification of areas that would serve as good antigens for thedevelopment of antibodies for use against specific amino acid sequencesof the antigen. Such sequences may be at different regions such asoutside, membrane or inside of the PSM antigen. The development of thesespecific antibodies would provide for immunohistochemical identificationof the antigen. These derived antibodies could then be developed foruse, especially ones that work in paraffin fixed sections as well asfrozen section as they have the greatest utility for immunodiagnosis.

3. Restriction Fragment Length Polymorphism and Genomic DNA

Restriction fragment length polymorphisms (RFLPS) have proven to beuseful in documenting the progression of genetic damage that occursduring tumor initiation and promotion. It may be that RFLP analysis willdemonstrate that changes in PSM sequence restriction mapping may provideevidence of predisposition to risk or malignant potential or progressionof the prostatic tumor.

Depending on the chromosomal location of the PSM antigen, the PSMantigen gene may serve as a useful chromosome location marker forchromosome analysis.

4. Serum

With the development of antigen specific antibodies, if the antigen orselected antigen fragments appear in the serum they may provide for aserum marker for the presence of metastatic disease and be usefulindividually or in combination with other prostate specific markers.

5. Imaging

As the cDNA sequence implies that the antigen has the characteristics ofa membrane spanning protein with the majority of the protein on theexofacial surface, antibodies, especially monoclonal antibodies to thepeptide fragments exposed and specific to the tumor may provide fortumor imaging local extension of metastatic tumor or residual tumorfollowing prostatectomy or irradiation. The knowledge of the codingregion permits the generation of monoclonal antibodies and these can beused in combination to provide for maximal imaging purposes. Because theantigen shares a similarity with the transferrin receptor based on cDNAanalysis (approximately 54%), it may be that there is a specific normalligand for this antigen and that identification of the ligand(s) wouldprovide another means of imaging.

6. Isolation of Ligands

The PSM antigen can be used to isolate the normal ligand(s) that bind toit. These ligand(s) depending on specificity may be used for targeting,or their serum levels may be predictive of disease status. If it isfound that the normal ligand for PSM is a carrier molecule then it maybe that PSM could be used to bind to that ligand for therapy purposes(like an iron chelating substance) to help remove the ligand from thecirculation. If the ligand promotes tumor growth or metastasis thenproviding soluble PSM antigen would remove the ligand from binding theprostate. Knowledge of PSM antigen structure could lend to generation ofsmall fragment that binds ligand which could serve the same purpose.

7. Therapeutic Uses

a) Ligands. The knowledge that the cDNA structure of PSM antigen sharesstructural homology; with the transferrin receptor (54% on the nucleicacid level) implies that there may be an endogenous ligand for thereceptor that may or may not be transferrin-like. Transferrin is thoughtto be a ligand that transports iron into the cell after binding to thetransferrin receptor. However, apotransferrin is being reported to be agrowth factor for some cells which express the transferrin receptor(30). Whether transferrin is a ligand for this antigen or some otherligand binds to this ligand remains to be determined. If a ligand isidentified it may carry a specific substance such as a metal ion (ironor zinc or other) into the tumor and thus serve as a means to delivertoxic substances (radioactive or cytotoxic chemical i.e. toxin likericin or cytotoxic alkylating agent or cytotoxic prodrug) to the tumor.

The main metastatic site for prostatic tumor is the bone. The bone andbone stroma are rich in transferrin. Recent studies suggest that thismicroenvironment is what provides the right “soil” for prostaticmetastasis in the bone (31). It may be that this also promotesattachment as well, these factors which reduce this ability may diminishprostatic metastasis to the bone and prostatic metastatic growth in thebone.

It was found that the ligand for the new antigen (thought to be anoncogene and marker of malignant phenotype in breast carcinoma) servedto induce differentiation of breast cancer cells and thus could serve asa treatment for rather than promotor of the disease. It may be thatligand binding to the right region of PSM whether with natural ligand orwith an antibody may serve a similar function.

Antibodies against PSM antigen coupled with a cytotoxic agent will beuseful to eliminate prostate cancer cells. Transferrin receptorantibodies with toxin conjugates are cytotoxic to a number of tumorcells as tumor cells tend to express increased levels of transferrinreceptor (32). Transferrin receptors take up molecules into the cell byendocytosis. Antibody drug combinations can be toxic. Transferrin linkedtoxin can be toxic.

b) Antibodies against PSM antigen coupled with a cytotoxic agent will beuseful to eliminate prostate cancer cells. The cytotoxic agent may be aradioisotope or toxin as known in ordinary skill of the art. The linkageof the antibody and the toxin or radioisotope can be chemical. Examplesof direct linked toxins are doxorubicin, chlorambucil, ricin,pseudomonas exotoxin etc., or a hybrid toxin can be generated ½ withspecificity for PSM and the other ½ with specificity for the toxin. Sucha bivalent molecule can serve to bind to the tumor and the other ½ todeliver a cytotoxic to the tumor or to bind to and activate a cytotoxiclymphocyte such as binding to the T₁-T₃ receptor complex. Antibodies ofrequired specificity can also be cloned into T cells and by replacingthe immunoglobulin domain of the T cell receptor (TcR); cloning in thedesired MAb heavy and light chains; splicing the U_(h) and U_(L) genesegments with the constant regions of the α and β TCR chains andtransfecting these chimeric Ab/TcR genes in the patients' T cells,propagating these hybrid cells and infusing them into the patient (33).Specific knowledge of tissue specific antigens for targets andgeneration of MAb's specific for such targets will help make this ausable approach. Because the PSM antigen coding region providesknowledge of the entire coding region, it is possible to generate anumber of antibodies which could then be used in combination to achievean additive or synergistic anti-tumor action. The antibodies can belinked to enzymes which can activate non-toxic prodrugs at its site ofthe tumor such as Ab-carboxypeptidase and 4-(bis(2chloroethyl)amino)benzoyl-α-glutamic acid and its active parent drug inmice (34).

It is possible to produce a toxic genetic chimera such as TP-40 agenetic recombinant that possesses the cDNA from TGF-alpha and the toxicportion of pseudomonas exotoxin so the TGF and portion of the hybridbinds the epidermal growth factor receptor (EGFR) and the pseudomonasportion gets taken up into the cell enzymatically and inactivates theribosomes ability to perform protein synthesis resulting in cell death.

In addition, once the ligand for the PSM antigen is identified, toxincan be chemically conjugated to the ligands. Such conjugated ligands canbe therapeutically useful. Examples of the toxins are daunomycin,chlorambucil, ricin, pseudomonas exotoxin, etc. Alternatively, chimericconstruct can be created linking the cDNA of the ligand with the cDNA ofthe toxin. An example of such toxin is TGFα and pseudomonas exotoxin(35)

8. Others

The PSM antigen may have other uses. It is well known that the prostateis rich in zinc, if the antigen provides function relative to this orother biologic function the PSM antigen may provide for utility in thetreatment of other prostatic pathologies such as benign hyperplasticgrowth and/or prostatitis.

Because purified PSM antigen can be generated, the purified PSM antigencan be linked to beads and use it like a standard “affinity”purification. Serum, urine or other biological samples can be used toincubate with the PSM antigen bound onto beads. The beads may be washedthoroughly and then eluted with salt or pH gradient. The eluted materialis SDS gel purified and used as a sample for microsequencing. Thesequences will be compared with other known proteins and if unique, thetechnique of degenerated PCR can be employed for obtaining the ligand.Once known, the affinity of the ligand will be determined by standardprotocols (15).

References of Example 1

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(1986) Tumor invasion and metastases: role of the    extracellular matrix. Cancer Res. 46:1-7.-   8. Horoszewicz, J. S., et al. (1987) Monoclonal antibodies to a new    antigenic marker in epithelial prostatic cells and serum of    prostatic cancer patients. Anticancer Res. 7:927-936.-   9. Horoszewicz, J. S., et al. (1983) LNCaP model of human prostatic    carcinoma. Cancer Res., 43:1809-1818.-   10. Lopes, D., et al. (1990) Immunohistochemical and pharmacokinetic    characterization of the site-specific immunoconjugate CYT-356,    derived from anti-prostate monoclonal antibody 7E11-C5. Cancer Res.,    50:6423-6429.-   11. Wright, Jr., et al., (1990) Characterization of a new carcinoma    associated marker:7E11-C5. Antibod. Immunoconj.    Radiopharm.3:(abst#193).-   12. Feng, Q., et al., (1991) Purification and biochemical    characterization of the 7E11-C5 prostate carcinoma associated    antigen. Proc. Amer. Assoc. Cancer Res. 32:239.-   13. Axelrod, H. R., et al., Preclinical results and human    immunohistochemical studies with ⁹⁰Y-CYT-356. A New prostate cancer    agent. Abstract 596. AUA 87th Annual. Meeting, May 10-14, 1992.    Washington, D.C.-   14. Maniatis, T., et al., (1982) Molecular Cloning; Cold Spring    Harbor Laboratory, pp. 197-98 (1982).-   15. Maniatis, et al., (1982) Molecular Cloning, Cold Spring Harbor    Laboratory.-   16. Methods in Enzymology vol. 34: 1-810, 1974 (E) B. Jacoby and M.    Wilchek Academic Press, New York 1974.-   17. Hogan B. et al. (1986) Manipulating the Mouse Embryo, A    Laboratory Manual, Cold Spring Harbor Laboratory.-   18. Capecchi M. R. Science (1989) 244:1288-1292; Zimmer, A. and    Gruss, P. (1989) Nature 338:150-153.-   19. Trowbridge , I. S., (1982) Prospects for the clinical use of    cytotoxic monoclonal antibodies conjugates in the treatment of    cancer. Cancer Surveys 1:543-556.-   20. Hank, S. K. (1987) Homology probing: Identification of cDNA    clones encoding members of the protein-serine kinase family. Proc.    Natl. Acad. Sci. 84:388-392.-   21. Lee, C. C., et al., (1988) Generation of cDNA probes directed by    amino acid sequences: cloning of urate oxidase. Science, 239, 1288.-   22. Girgis, S. I., et al. (1988) Generation of DNA probes for    peptides with highly degenerate codons using mixed primer PCR.    Nucleic Acids Res. 16:10932.-   23. Kartner, N., et al. (1977) Isolation of plasma membranes from    human skin fibroblasts. J. Membrane Biology, 36:191-211.-   24. Hsu, S. M., et al. (1981) Comparative study of the    immunoperoxidase, anti-peroxidase, and avidin-biotin complex method    for studying polypeptide hormones with radioimmunoassay antibodies.    Am. J. Pathology, 75:734.-   25. Tempst, P., et al. (1989) Examination of automated polypeptide    sequencing using standard phenylisothiocyanate reagent and    subpicomole high performance liquid chromatography analysis.    Analytical Biochem. 183:290-300.-   26. Birnboim, H. C. (1983) A rapid alkaline extraction method for    the isolation of plasmid DNA. Meth. Enzymol, 100:243-255.-   27. Sanger, F., et al. (1977) DNA sequencing with chain-terminating    inhibitors. Proc. Natl. Acad. Sci. USA, 74:5463-5467.-   28. Grunstein, M., et al. (1975) Colony hybridization as a method    for the isolation of cloned DNAs that contain a specific gene. Proc.    Natl. Acad. Sci. USA, 72:3961.-   29. Feinberg, A. P., et al. (1983) A technique for radiolabeling DNA    restriction endonuclease fragments to high specific activity. Anal.    Biochem, 132, 6.-   30. Rave, N., et al. (1979) Identification of procollagen mRNAs    transferred to diazobenzylomethyl paper from formaldehyde gels.    Nucleic Acids Research, 6:3559.-   31. Voeller, H. J., et al. (1991) v-ras^(H) expression confers    hormone-independent in-vitro growth to LNCaP prostate carcinoma    cells. Molec. Endocrinology. Vol. 5. No. 2, 209-216.-   32. Sirbasku, D. A. (1991) Purification of an equine apotransferrin    variant (thyromedin) essential for thyroid hormone dependent growth    of GH₁, rat pituitary tumor cells in chemically defined culture.    Biochem., 30:295-301.-   33. Rossi, M. C. (1992) Selective stimulation of prostatic carcinoma    cell proliferation by transferrin. Proc. Natl. Acad. Sci. (USA)    89:6197-6201.-   34. Eshhan, Z. (1990) Chimeric T cell receptor which incorporates    the anti-tumor specificity of a monoclonal antibody with the    cytolytic activity of T cells: a model system for immunotherapeutic    approach. B. J. Cancer 62:27-29.-   35. Antonie, P. (1990) Disposition of the prodrug 4-(bis(2    chloroethyl)amino)benzoyl-α-glutamic acid and its active parent in    mice. B. J. Cancer 62:905-914.-   36. Heimbrook, D. C., et al. (1990) Transforming growth factor    alpha-pseudomonas exotoxin fusion protein prolongs survival of nude    mice bearing tumor xenografts. Proc. Natl. Acad. Sci. (USA)    87:4697-4701.-   37. Chiarodo, A. National Cancer Institute roundtable on prostate    cancer; future research directions. Cancer Res., 51: 2498-2505,    1991.-   38. Abdel-Nabi, H., Wright, G. L., Gulfo, J. V., Petrylak, D. P.,    Neal, C. E., Texter, J. E., Begun, F. P., Tyson, I., Heal, A.,    Mitchell, E., Purnell, G., and Harwood, S. J. Monoclonal antibodies    and radioimmunoconjugates in the diagnosis and treatment of prostate    cancer. Semin. Urol., 10: 45-54, 1992.

Example 2 Expression of the Prostate Specific Membrane Antigen

A 2.65 kb complementary DNA encoding PSM was cloned. Immunohistochemicalanalysis of the LNCaP, DU-145, and PC-3 prostate cancer cell lines forPSM expression using the 7E11-C5.3 antibody reveals intense staining inthe LNCaP cells, with no detectable expression in both the DU-145 andPC-3-cells. Coupled in-vitro transcription/translation of the 2.65 kbfull-length PSM cDNA yields an 84 kDa protein corresponding to thepredicted polypeptide molecular weight of PSM. Post-translationalmodification of this protein with pancreatic canine microsomes yieldsthe expected 100 kDa PSM antigen. Following transfection of PC-3 cellswith the full-length PSM cDNA in a eukaryotic expression vectorapplicant's detect expression of the PSM glycoprotein by Westernanalysis using the 7E11-C5.3 monoclonal antibody. Ribonucleaseprotection analysis demonstrates that the expression of PSM mRNA isalmost entirely prostate-specific in human tissues. PSM expressionappears to be highest in hormone-deprived states and is hormonallymodulated by steroids, with DHT downregulating PSM expression in thehuman prostate cancer cell line LNCaP by 8-10 fold, testosteronedownregulating PSM by 3-4 fold, and corticosteroids showing nosignificant effect. Normal and malignant prostatic tissues consistentlyshow high PSM expression, whereas heterogeneous, and at times absent,from expression of PSM in benign prostatic hyperplasia. LNCaP tumorsimplanted and grown both orthotopically and subcutaneously in nude mice,abundantly express PSM providing an excellent in-vivo model system tostudy the regulation and modulation of PSM expression.

Materials and Methods:

Cells and Reagents:

The LNCaP, DU-145, and PC-3 cell lines were obtained from the AmericanType Culture Collection. Details regarding the establishment andcharacteristics of these cell lines have been previously published(5A,7A,8A). Unless specified otherwise, LNCaP cells were grown in RPMI1640 media supplemented with L-glutamine, nonessential amino acids, and5% fetal calf serum (Gibco-BRL, Gaithersburg, Md.) in a CO₂ incubator at37C. DU-145 and PC-3 cells were grown in minimal essential mediumsupplemented with 10% fetal calf serum. All cell media were obtainedfrom the MSKCC Media Preparation Facility. Restriction and modifyingenzymes were purchased from Gibco-BRL unless otherwise specified.

Immunohistochemical Detection of PSM:

Avidin-biotin method of detection was employed to analyze prostatecancer cell lines for PSM antigen expression (9A). Cell cytospins weremade on glass slides using 5×10⁴ cells/100 ul per slide. Slides werewashed twice with PBS and then incubated with the appropriate suppressorserum for 20 minutes. The suppressor serum was drained off and the cellswere incubated with diluted 7E11-C5.3 (5 g/ml) monoclonal antibody for 1hour. Samples were then washed with PBS and sequentially incubated withsecondary antibodies for 30 minutes and with avidin-biotin complexes for30 minutes. Diaminobenzidine served as the chromogen and colordevelopment followed by hematoxylin counterstaining and mounting.Duplicate cell cytospins were used as controls for each experiment. As apositive control, the anti-cytokeratin monoclonal antibody CAM 5.2 wasused following the same procedure described above. Human EJ bladdercarcinoma cells served as a negative control.

In-Vitro Transcription/Translation of PSM Antigen:

Plasmid 55A containing the full length 2.65 kb PSM cDNA in the plasmidpSPORT 1 (Gibco-BRL) was transcribed in-vitro using the Promega TNTsystem (Promega Corp. Madison, Wis.). T7 RNA polymerase was added to thecDNA in a reaction mixture containing rabbit reticulocyte lysate, anamino acid mixture lacking methionine, buffer, and ³⁵S-Methionine(Amersham) and incubated at 30 C for 90 minutes. Post-translationalmodification of the resulting protein was accomplished by the additionof pancreatic canine microsomes into the reaction mixture (Promega Corp.Madison, Wis.). Protein products were analyzed by electrophoresis on 10%SDS-PAGE gels which were subsequently treated with Amplifyautoradiography enhancer (Amersham, Arlington Heights, Ill.) accordingto the manufacturers instructions and dried at 80 C in a vacuum dryer.Gels were autoradiographed overnight at −70 C using Hyperfilm MP(Amersham).

Transfection of PSM into PC-3 Cells:

The full length PSM cDNA was subcloned into the pREP7 eukaryoticexpression vector (Invitrogen, San Diego, Calif.). Plasmid DNA waspurified from transformed DH5-alpha bacteria (Gibco-BRL) using Qiagenmaxi-prep plasmid isolation columns (Qiagen Inc., Chatsworth, Calif.).Purified plasmid DNA (6-10 g) was diluted with 900 ul of Optimem media(Gibco-BRL) and mixed with 30 ul of Lipofectin reagent (Gibco-BRL) whichhad been previously diluted with 900 l of Optimem media. This mixturewas added to T-75 flasks of 40-50% confluent PC-3 cells in Optimemmedia. After 24-36 hours, cells were trypsinized and split into 100 mmdishes containing RPMI 1640 media supplemented with 10% fetal calf serumand 1 mg/ml of Hygromycin B (Calbiochem, La Jolla, Calif.). The dose ofHygromycin B used was previously determined by a time course/doseresponse cytotoxicity assay. Cells were maintained in this media for 2-3weeks with changes of media and Hygromycin B every 4-5 days untildiscrete colonies appeared. Colonies were isolated using 6 mm cloningcylinders and expanded in the same media. As a control, PC-3 cells werealso transfected with the pREP7 plasmid alone. RNA was isolated from thetransfected cells and PSM mRNA expression was detected by both RNaseProtection analysis (described later) and by Northern analysis.

Western Blot Detection of PSM Expression:

Crude protein lysates were isolated from LNCaP, PC-3, andPSM-transfected PC-3 cells as previously described (10A). LNCaP cellmembranes were also isolated according to published methods (10A).Protein concentrations were quantitated by the Bradford method using theBioRad protein reagent kit (BioRad, Richmond, Calif.). Followingdenaturation, 20 μg of protein was electrophoresed on a 10% SDS-PAGE gelat 25 mA for 4 hours. Gels were electroblotted onto Immobilon Pmembranes (Millipore, Bedford, Mass.) overnight at 4 C. Membranes wereblocked in 0.15M NaCl/0.01M Tris-HCl (TS) plus 5% BSA followed by a 1hour incubation with 7E11-C5.3 monoclonal antibody (10 μg/ml). Blotswere washed 4 times with 0.15M NaCl/0.01M Tris-HCl/0.05% Triton-X 100(TS-X) and incubated for 1 hour with rabbit anti-mouse IgG (AccurateScientific, Westbury, N.Y.) at a concentration of 10 μg/ml.

Blots were then washed 4 times with TS-X and labeled with ¹²⁵I-Protein A(Amersham, Arlington Heights, Ill.) at a concentration of 1 millioncpm/ml. Blots were then washed 4 times with TS-X and dried on Whatman3MM paper, followed by overnight autoradiography at −70 C usingHyperfilm MP (Amersham).

Orthotopic and Subcutaneous LNCaP Tumor Growth in Nude Mice:

LNCaP cells were harvested from sub-confluent cultures by a one minuteexposure to a solution of 0.25% trypsin and 0.02% EDTA. Cells wereresuspended in RPMI 1640 media with 5% fetal bovine serum, washed anddiluted in either Matrigel (Collaborative Biomedical Products, Bedford,Mass.) or calcium and magnesium-free Hank's balanced salt solution(HBSS). Only single cell suspensions with greater than 90% viability bytrypan blue exclusion were used for in vivo injection. Male athymicSwiss (nu/nu) nude mice 4-6 weeks of age were obtained from the MemorialSloan-Kettering Cancer Center Animal Facility. For subcutaneous tumorcell injection one million LNCaP cells resuspended in 0.2 mls. ofMatrigel were injected into the hindlimb of each mouse using adisposable syringe fitted with a 28 gauge needle. For orthotopicinjection, mice were first anesthetized with an intraperitonealinjection of Pentobarbital and placed in the supine position. Theabdomen was cleansed with Betadine and the prostate was exposed througha midline incision. 2.5 million LNCaP tumor cells in 0.1 ml. wereinjected directly into either posterior lobe using a 1 ml disposablesyringe and a 28 gauge needle. LNCaP cells with and without Matrigelwere injected. Abdominal closure was achieved in one layer usingAutoclip wound clips (Clay Adams, Parsippany, N.J.). Tumors wereharvested in 6-8 weeks, confirmed histologically by faculty of theMemorial Sloan-Kettering Cancer Center Pathology Department, and frozenin liquid nitrogen for subsequent RNA isolation.

RNA Isolation:

Total cellular RNA was isolated from cells and tissues by standardtechniques (11,12) as well as by using RNAzol B (Cinna/Biotecx, Houston,Tex.). RNA concentrations and quality were assessed by UV spectroscopyon a Beckman DU 640 spectrophotometer and by gel analysis. Human tissuetotal RNA samples were purchased from Clontech Laboratories, Inc., PaloAlto, Calif.

Ribonuclease Protection Assays:

A portion of the PSM cDNA was subcloned into the plasmid vector pSPORT 1(Gibco-BRL) and the orientation of the cDNA insert relative to theflanking T7 and SP6 RNA polymerase promoters was verified by restrictionanalysis. Linearization of this plasmid upstream of the PSM insertfollowed by transcription with SP6 RNA polymerase yields a 400nucleotide antisense RNA probe, of which 350 nucleotides should beprotected from RNase digestion by PSM RNA. This probe was used in FIG.20. Plasmid IN-20, containing a 1 kb partial PSM cDNA in the plasmid pCRII (Invitrogen) was also used for riboprobe synthesis. IN-20 linearizedwith Xmn I (Gibco-BRL) yields a 298 nucleotide anti-sense RNA probe whentranscribed using SP6 RNA polymerase, of which 260 nucleotides should beprotected from RNase digestion by PSM mRNA. This probe was used in FIGS.21 and 22. Probes were synthesized using SP6 RNA polymerase (Gibco-BRL),rNTPs (Gibco-BRL), RNAsin (Promega), and ³²P-rCTP (NEN, Wilmington,Del.) according to published protocols (13). Probes were purified overNENSORB 20 purification columns (NEN) and approximately 1 million cpm ofpurified, radiolabeled PSM probe was mixed with 10μ of each RNA andhybridized overnight at 45 C using buffers and reagents from the RPA IIkit (Ambion, Austin, Tex.). Samples were processed as per manufacturer'sinstructions and analyzed on 5% polyacrilamide/7M urea denaturing gelsusing Seq ACRYL reagents (ISS, Natick, Mass.). Gels were pre-heated to55 C and run for approximately 1-2 hours at 25 watts. Gels were thenfixed for 30 minutes in 10% methanol/10% acetic acid, dried onto Whatman3MM paper at 80 C in a BioRad vacuum dryer and autoradiographedovernight with Hyperfilm MP (Amersham). Quantitation of PSM expressionwas determined by using a scanning laser densitometer (LKB, Piscataway,N.J.).

Steroid Modulation Exeriment:

LNCaP cells (2 million) were plated onto T-75 flasks in RPMI 1640 mediasupplemented with 5% fetal calf serum and grown 24 hours untilapproximately 30-40% confluent. Flasks were then washed several timeswith phophate-buffered saline and RPMI medium supplemented with 5%charcoal-extracted serum was added. Cells were then grown for another 24hours, at which time dihydrotesterone, testosterone, estradiol,progesterone, and dexamethasone (Steraloids Inc., Wilton, N.H.) wereadded at a final concentration of 2 nM. Cells were grown for another 24hours and RNA was then harvested as previously described and PSMexpression analyzed by ribonuclease protection analysis.

Experimental Results

Immuohistochemical Detection of PSM:

Using the 7E11-C5.3 anti-PSM monoclonal antibody, PSM expression isclearly detectable in the LNCaP prostate cancer cell line, but not inthe PC-3 and DU-145 cell lines (FIGS. 17A-17C). All normal and malignantprostatic tissues analyzed stained positively for PSM expression.

In-Vitro Transcription/Translation of PSM Antigen:

As shown in FIG. 18, coupled in-vitro transcription/translation of the2.65 kb full-length PSM cDNA yields an 84 kDa protein species inagreement with the expected protein product from the 750 amino acid PSMopen reading frame. Following post-translational modification usingpancreatic canine microsomes were obtained a 100 kDa glycosylatedprotein species consistent with the mature, native PSM antigen.

Detection of PSM Antigen in LaNCaP Cell Membranes and Transfected PC-3Cells:

PC-3 cells transfected with the full length PSM cDNA in the pREP7expression vector were assayed for expression of SM mRNA by Northernanalysis. A clone with high PSM mRNA expression was selected for PSMantigen analysis by Western blotting using the 7E11-C5.3 antibody. InFIG. 19, the 100 kDa PSM antigen is well expressed in LNCaP cell lysateand membrane fractions, as well as in PSM-transfected PC-3 cells but notin native PC-3 cells. This detectable expression in the transfected PC-3cells proves that the previously cloned 2.65 kb PSM cDNA encodes theantigen recognized by the 7E11-C5.3 anti-prostate monoclonal antibody.

PSM mRNA Expression:

Expression of PSM mRNA in normal human tissues was analyzed usingribonuclease protection assays. Tissue expression of PSM appearspredominantly within the prostate, with very low levels of expressiondetectable in human brain and salivary gland (FIG. 20). No detectablePSM mRNA expression was evident in non-prostatic human tissues whenanalyzed by Northern analysis. On occasion it is noted that detectablePSM expression in normal human small intestine tissue, however this mRNAexpression is variable depending upon the specific riboprobe used. Allsamples of normal human prostate and human prostatic adenocarcinomaassayed have revealed clearly detectable PSM expression, whereasgenerally decreased or absent expression of PSM in tissues exhibitingbenign hyperplasia (FIG. 21). In human LNCaP tumors grown bothorthotopically and subcutaneously in nude mice abundant PSM expressionwith or without the use of matrigel, which is required for the growth ofsubcutaneously implanted LNCaP cells was detected (FIG. 21). PSM mRNAexpression is distinctly modulated by the presence of steroids inphysiologic doses (FIG. 22). DHT downregulated expression by 8-10 foldafter 24 hours and testosterone diminished PSM expression by 3-4 fold.Estradiol and progesterone also downregulated PSM expression in LNCaPcells, perhaps as a result of binding to the mutated androgen receptorknown to exist in the LNCaP cell. Overall, PSM expression is highest inthe untreated LNCaP cells grown in steroid-depleted media, a situationthat simulates the hormone-deprived (castrate) state in-vivo. Thisexperiment was repeated at steroid dosages ranging from 2-200 nM and attime points from 6 hours to 7 days with similar results; maximaldownregulation of PSM mRNA was seen with DHT at 24 hours at doses of2-20 nM.

Experimental Discussion

Previous research has provided two valuable prostatic bio-markers, PAPand PSA, both of which have had a significant impact on the diagnosis,treatment, and management of prostate malignancies. The present workdescribing the preliminary characterization of the prostate-specificmembrane antigen (PSM) reveals it to be a gene with many interestingfeatures. PSM is almost entirely prostate-specific as are PAP and PSA,and as such may enable further delineation of the unique functions andbehavior of the prostate. The predicted sequence of the PSM protein (3)and its presence in the LNCaP cell membrane as determined by Westernblotting and immunohistochemistry, indicate that it is an integralmembrane protein. Thus, PSM provides an attractive cell surface epitopefor antibody-directed diagnostic imaging and cytotoxic targetingmodalities (14). The ability to synthesize the PSM antigen in-vitro andto produce tumor xenografts maintaining high levels of PSM expressionprovides us with a convenient and attractive model system to furtherstudy and characterize the regulation and modulation of PSM expression.Also, the high level of PSM expression in the LNCaP cells provides anexcellent in-vitro model system. Since PSM expression ishormonally-responsive to steroids and may be highly expressed inhormone-refractory disease (15). The detection of PSM mRNA expression inminute quantities in brain, salivary gland, and small intestine warrantsfurther investigation, although these tissues were negative forexpression of PSM antigen by immunohistochemistry using the 7E11-C5.3antibody (16). In all of these tissues, particularly small intestine,mRNA expression using a probe corresponding to a region of the PSM cDNAnear the 3′ end, whereas expression when using a 5′ end PSM probe wasnot detected. These results may indicate that the PSM mRNA transcriptundergoes alternative splicing in different tissues.

Applicants approach is based on prostate tissue specific promotor:enzyme or cytokine chimeras. Promotor specific activation of prodrugssuch as non toxic gancyclovir which is converted to a toxic metaboliteby herpes simplex thymidine kinase or the prodrug4-(bis(2chloroethyl)amino)benzoyl-1-glutamic acid to the benzoic acidmustard alkylating agent by the pseudomonas carboxy peptidase G2 wasexamined. As these drugs are activated by the enzyme (chimera)specifically in the tumor the active drug is released only locally inthe tumor environment, destroying the surrounding tumor cells. Promotorspecific activation of cytokines such as IL-12, IL-2 or GM-CSF foractivation and specific antitumor vaccination is examined. Lastly, thetissue specific promotor activation of cellular death genes may alsoprove to be useful in this area.

Gene Therapy Chimeras:

The establishment of “chimeric DNA” for gene therapy requires thejoining of different segments of DNA together to make a new DNA that hascharacteristics of both precursor DNA species involved in the linkage.In this proposal the two pieces being linked involve differentfunctional aspects of DNA, the promotor region which allows for thereading of the DNA for the formation of mRNA will provide specificityand the DNA sequence coding for the mRNA will provide for therapeuticfunctional DNA.

DNA-Specified Enzyme or Cytokine mRNA:

When effective, antitumor drugs can cause the regression of very largeamounts of tumor. The main requirements for antitumor drug activity isthe requirement to achieve both a long enough time (t) and high enoughconcentration (c) (cxt) of exposure of the tumor to the toxic drug toassure sufficient cell damage for cell death to occur. The drug alsomust be “active” and the toxicity for the tumor greater than for thehosts normal cells (22). The availability of the drug to the tumordepends on tumor blood flow and the drugs diffusion ability. Blood flowto the tumor does not provide for selectivity as blood flow to manynormal tissues is often as great or greater than that to the tumor. Themajority of chemotherapeutic cytotoxic drugs are often as toxic tonormal tissue as to tumor tissue. Dividing cells are often moresensitive than non-dividing normal cells, but in many slow growing solidtumors such as prostatic cancer this does not provide for antitumorspecificity (22).

Previously a means to increase tumor specificity of antitumor drugs wasto utilize tumor associated enzymes to activate nontoxic prodrugs tocytotoxic agents (19). A problem with this approach was that most of theenzymes found in tumors were not totally specific in their activity andsimilar substrate active enzymes or the same enzyme at only slightlylower amounts was found in other tissue and thus normal tissues werestill at risk for damage.

To provide absolute specificity and unique activity, viral, bacterialand fungal enzymes which have unique specificity for selected prodrugswere found which were not present in human or other animal cells.Attempts to utilize enzymes such as herpes simplex thymidine kinase,bacterial cytosine deaminase and carboxypeptidase G-2 were linked toantibody targeting systems with modest success (19). Unfortunately,antibody targeted enzymes limit the number of enzymes available percell. Also, most antibodies do not have a high tumor target to normaltissue ratio thus normal tissues are still exposed reducing thespecificity of these unique enzymes. Antibodies are large molecules thathave poor diffusion properties and the addition of the enzymes molecularweight further reduces the antibodies diffusion.

Gene therapy could produce the best desired result if it could achievethe specific expression of a protein in the tumor and not normal tissuein order that a high local concentration of the enzyme be available forthe production in the tumor environment of active drug (21).

Cytokines:

Results demonstrated that tumors such as the bladder and prostate werenot immunogenic, that is the administration of irradiated tumor cells tothe animal prior to subsequent administration of non-irradiated tumorcells did not result in a reduction of either the number of tumor cellsto produce a tumor nor did it reduce the growth rate of the tumor. Butif the tumor was transfected with a retrovirus and secreted largeconcentrations of cytokines such as Il-2 then this could act as anantitumor vaccine and could also reduce the growth potential of analready established and growing tumor. IL-2 was the best, GM-CSF alsohad activity whereas a number of other cytokines were much less active.In clinical studies just using IL-2 for immunostimulation, very largeconcentrations had to be given which proved to be toxic. The key to thesuccess of the cytokine gene modified tumor cell is that the cytokine isproduced at the tumor site locally and is not toxic and that itstimulates immune recognition of the tumor and allows specific and nontoxic recognition and destruction of the tumor. The exact mechanisms ofhow IL-2 production by the tumor cell activates immune recognition isnot fully understood, but one explanation is that it bypasses the needfor cytokine production by helper T cells and directly stimulates tumorantigen activated cytotoxic CD8 cells. Activation of antigen presentingcells may also occur.

Tissue Promotor-Specific Chimera DNA Activation

Non-Prostatic Tumor Systems:

It has been observed in non-prostatic tumors that the use of promotorspecific activation can selectively lead to tissue specific geneexpression of the transfected gene. In melanoma the use of thetyrosinase promotor which codes for the enzyme responsible for melaninexpression produced over a 50 fold greater expression of the promotordriven reporter gene expression in melanoma cells and not non melanomacells. Similar specific activation was seen in the melanoma cellstransfected when they were growing in mice. In that experiment nonon-melanoma or melanocyte cell expressed the tyrosinase drive reportergene product. The research group at Welcome Laboratories have cloned andsequenced the promoter region of the gene coding for carcinoembryonicantigen (CEA). CEA is expressed on colon and colon carcinoma cells butspecifically on metastatic. A gene chimera was generated which cytosinedeaminase. Cytosine deaminase which converts 5 flurorocytosine into 5fluorouracil and observed a large increase in the ability to selectivelykill CEA promotor driven colon tumor cells but not normal liver cells.In vivo they observed that bystander tumor cells which were nottransfected with the cytosine deaminase gene were also killed, and thatthere was no toxicity to the host animal as the large tumors wereregressing following treatment. Herpes simplex virus, (HSV), thymidinekinase similarly activates the prodrug gancyclovir to be toxic towardsdividing cancer cells and HSV thymidine kinase has been shown to bespecifically activatable by tissue specific promoters.

Prostatic Tumor Systems:

The therapeutic key to effective cancer therapy is to achievespecificity and spare the patient toxicity. Gene therapy may provide akey part to specificity in that non-essential tissues such as theprostate and prostatic tumors produce tissue specific proteins, such asacid phosphatase (PAP), prostate specific antigen (PSA), and a genewhich was cloned, prostate-specific membrane antigen (PSM). Tissues suchas the prostate contain selected tissue specific transcription factorswhich are responsible for binding to the promoter region of the DNA ofthese tissue specific mRNA. The promoter for PSA has been cloned.Usually patients who are being treated for metastatic prostatic cancerhave been put on androgen deprivation therapy which dramatically reducesthe expression of mRNA for PSA. PSM on the other hand increases inexpression with hormone deprivation which-means it would be even moreintensely expressed on patients being treated with hormone therapy.

References of Example 2

-   1. Coffey, D. S. Prostate Cancer—An overview of an increasing    dilemma. Cancer Supplement, 71, 3: 880-886, 1993.-   2. Chiarodo, A. National Cancer Institute roundtable on prostate    cancer; future research directions. Cancer Res., 51: 2498-2505,    1991.-   3. Israeli, R. S., Powell, C. T., Fair, W. R., and Heston, W. D. W.    Molecular cloning of a complementary DNA encoding a    prostate-specific membrane antigen. Cancer Res., 53: 227-230, 1993.-   4. Horoszewicz, J. S., Kawinski, E., and Murphy, G. P. Monoclonal    antibodies to a new antigenic marker in epithelial cells and serum    of prostatic cancer patients. Anticancer Res., 7: 927-936, 1987.-   5. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P.,    Rosenthal, H., Chu, T. M., Mirand, E. A., and Murphy, G. P. LNCaP    model of human prostatic carcinoma. Cancer Res., 43: 1809-1818,    1983.-   6. Abdel-Nabi, H., Wright, G. L., Gulfo, J. V., Petrylak, D. P.,    Neal, C. E., Texter, J. E., Begun, F. P., Tyson, I., Heal, A.,    Mitchell, E., Purnell, G., and Harwood, S. J. Monoclonal antibodies    and radioimmunoconjugates in the diagnosis and treatment of prostate    cancer. Semin. Urol., 10: 45-54, 1992.-   7. Stone, K. R., Mickey, D. D., Wunderli, H., Mickey, G. H., and    Paulson, D. F. Isolation of a human prostate carcinoma cell line    (DU-145). Int. J. Cancer, 21: 274-281, 1978.-   8. Kaign, M. E., Narayan, K. S., Ohnuki, Y., and Lechner, J. F.    Establishment and characterization of a human prostatic carcinoma    cell line (PC-3). Invest. Urol., 17: 16-23, 1979.-   9. Hsu, S. M., Raine, L., and Fanger, H. Review of present methods    of immunohistochemical detection. Am. J. Clin. Path. 75: 734-738,    1981.-   10. Harlow, E., and Lane, D. Antibodies: A Laboratory Manual. New    York: Cold Spring Harbor Laboratory, p. 449, 1988.-   11. Glisin, V., Crkvenjakov, R., and Byus, C. Ribonucleic acid    isolated by cesium chloride centrifugation. Biochemistry, 13:    2633-2637, 1974.-   12. Aviv, H., and Leder, P. Purification of biologically active    globin messenger RNA by chromatography on oligo-thymidylic acid    cellulose. Proc. Natl. Acad. Sci. USA, 69: 1408-1412, 1972.-   13. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T. A.,    Zinn, K., and Careen, M. R. Efficient in-vitro synthesis of    biologically active RNA and RNA hybridization probes from plasmids    containing a bacteriophage SP6 promoter. Nucl. Acids. Res. 12:    7035-7056, 1984.-   14.-   15. Axelrod, H. R., Gilman; S. C., D'Aleo, C. J., Petrylak, D.,    Reuter, V., Gulfo, J. V., Saad, A., Cordon-Cardo, C., and    Scher, H. I. Preclinical results and human immunohistochemical    studies with ⁹⁰Y-CYT-356; a new prostatic cancer therapeutic agent.    AUA Proceedings, Abstract 596, 1992.-   16. Lopes, A. D., Davis, W. L., Rosenstraus, M. J., Uveges, A. J.,    and Gilman, S. C. Immunohistochemical and pharmacokinetic    characterization of the site-specific immunoconjugate CYT-356    derived from antiprostate monoclonal antibody 7E11-C5. Cancer Res.,    50: 6423-6429, 1990.-   17. Troyer, J. K., Qi, F., Beckett, M. L., Morningstar, M. M., and    Wright, G. L. Molecular characterization of the 7E11-C5 prostate    tumor-associated antigen. AUA Proceedings. Abstract 482, 1993.-   18. Roemer, K., Friedmann, T. Concepts and strategies for human gene    therapy. FEBS. 223:212-225.-   19. Antonie, P. Springer, C. J., Bagshawe, F., Searle, F.,    Melton, R. G., Rogers, G. T., Burke, P. J., Sherwood, R. F.    Disposition of the prodrug    4-bis(2chloroethyl)amino)benzoyl-1-glutamic acid and its active    parentdrug in mice. Br. J. Cancer 62:909-914, 1990.-   20. Connor, J. Bannerji, R., Saito, S., Heston, W. D. W., Fair, W.    R., Gilboa, E. Regression of bladder tumors in mice treated with    interleukin 2 gene-modified tumor cells. J. Exp. Med.    177:1127-1134, 1993. (appendix)-   21. Vile R., Hart, I. R. In vitro and in vivo targeting of gene    expression to melanoma cells. Cancer Res. 53:962-967, 1993.-   22. Warner, J. A., Heston, W. D. W. Future developments of    nonhormonal systemic therapy for prostatic carcinoma. Urologic    Clinics of North America 18:25-33, 1991.-   23. Vile, R. G., Hart, I. R. Use of tissue specific expression of    the herpes simplex virus thymidine kinase gene to inhibit growth of    established murine melanomas following direct intratumoral injection    of DNA. Cancer Res. 53:3860-3864, 1993.

Example 3 Sensitive Detection of Prostatic Hematogenous HicrometastasesUsing PSA and PSM-Derived Primers in the Polymerase Chain Reaction

A PCR-based assay was developed enabling sensitive detection ofhematogenous micrometastases in patients with prostate cancer. “NestedPCR”, was performed by amplifying mRNA sequences unique toprostate-specific antigen and to the prostate-specific membrane antigen,and have compared their respective results. Micrometastases weredetected in 2/30 patients (6.7%) by PCR with PSA-derived primers, whilePSM-derived primers detected tumor cells in 19/16 patients (63.3%). All8 negative controls were negative with both PSA and PSM PCR. Assays wererepeated to confirm results, and PCR products were verified by DNAsequencing and Southern analysis. Patients harboring circulatingprostatic tumor cells as detected by PSM, and not by PSA-PCR included 4patients previously treated with radical prostatectomy and withnon-measurable serum PSA levels at the time of this assay. Thesignificance of these findings with respect to future disease recurrenceand progression will be investigated.

Improvement in the overall survival of patients with prostate cancerwill depend upon earlier diagnosis. Localized disease, without evidenceof extra-prostatic spread, is successfully treated with either radicalprostatectomy or external beam radiation, with excellent long-termresults (2,3). The major problem is that approximately two-thirds of mendiagnosed with prostate cancer already have evidence of advancedextra-prostatic spread at the time of diagnosis, for which there is atpresent no cure (4). The use of clinical serum markers such asprostate-specific antigen (PSA) and prostatic acid phosphatase (PAP)have enabled clinicians to detect prostatic carcinomas earlier andprovide useful parameters to follow responses to therapy (5). Yet,despite the advent of sensitive serum PSA assays, radionuclide bonescans, CT scans and other imaging modalities, results have not detectedthe presence of micrometastatic cells prior to their establishment ofsolid metastases. Previous work has been done utilizing the polymerasechain reaction to amplify mRNA sequences unique to breast, leukemia, andother malignant cells in the circulation and enable early detection ofmicrometastases (6,7). Recently, a PCR-based approach utilizing primersderived from the PSA DNA sequence was published (8). In this study 3/12patients with advanced, stage D prostate cancer had detectablehematogenous micrometastases.

PSM appears to be an integral membrane glycoprotein which is very highlyexpressed in prostatic tumors and metastases and is almost entirelyprostate-specific (10). Many anaplastic tumors and bone metastases havevariable and at times no detectable expression of PSA, whereas theselesions appear to consistently express high levels of PSM. Prostatictumor cells that escape from the prostate gland and enter thecirculation are likely to have the potential to form metastases and arepossibly the more aggressive and possibly anaplastic cells, a populationof cells that may not express high levels of PSA, but may retain highexpression of PSM. DNA primers derived from the sequences of both PSAand PSM in a PCR assay were used to detect micrometastatic cells in theperipheral circulation. Despite the high level of amplification andsensitivity of conventional RNA PCR, “Nested” PCR approach in which aamplified target sequence was employed, and subsequently use this PCRproduct as the template for another round of PCR amplification with anew set of primers totally contained within the sequence of the previousproduct. This approach has enabled us to increase the level of detectionfrom one prostatic tumor cell per 10,000 cells to better than one cellper ten million cells.

Materials and Methods

Cells and Reagents:

LNCaP and MCF-7 cells were obtained from the American Type CultureCollection (Rockville, Md.). Details regarding the establishment andcharacteristics of these cell lines have been previously published(11,12). Cells were grown in RPMI 1640 media supplemented withL-glutamine, nonessential amino acids, obtained from the MSKCC MediaPreparation Facility, and 5% fetal calf serum (Gibco-BRL, Gaithersburg,Md.) in a CO₂ incubator at 37 C. All cell media was obtained from theMSKCC Media Preparation Facility. Routine chemical reagents were of thehighest grade possible and were obtained from Sigma Chemical Company,St. Louis, Mo.

Patient Blood Specimens:

All blood specimens used in this study were from patients seen in theoutpatient offices of urologists on staff at MSKCC. Two anti-coagulated(purple top) tubes per patient were obtained at the time of theirregularly scheduled blood draws. Specimen procurement was conducted asper the approval of the MSKCC Institutional Review Board. Samples werepromptly brought to the laboratory for immediate processing. Serum PSAand PAP determinations were performed by standard techniques by theMSKCC Clinical Chemistry Laboratory. PSA determinations were performedusing the Tandem PSA assay (Hybritech, San Diego, Calif.). The eightblood specimens used as negative controls were from 2 males with normalserum PSA values and biopsy-proven BPH, one healthy female, 3 healthymales, one patient with bladder cancer, and one patient with acutepromyelocytic leukemia.

Blood Sample Processing/RNA Extraction:

4 ml of whole anticoagulated venous blood was mixed with 3 ml of icecold phosphate buffered saline and then carefully layered atop 8 ml ofFicoll (Pharmacia, Uppsala, Sweden) in a 15-ml polystyrene tube. Tubeswere centrifuged at 200×g for 30 min. at 4 C. Using a sterile pasteurpipette, the buffy coat layer (approx. 1 ml.) was carefully removed andrediluted up to 50 ml with ice cold phosphate buffered saline in a 50 mlpolypropylene tube. This tube was then centrifuged at 2000×g for 30 minat 4 C. The supernatant was carefully decanted and the pellet wasallowed to drip dry. One ml of RNazol B was then added to the pellet andtotal RNA was isolated as per manufacturers directions (Cinna/Biotecx,Houston, Tex.). RNA concentrations and purity were determined by UVspectroscopy on a Beckman DU 640 spectrophotometer and by gel analysis.

Determination of PCR Sensitivity:

RNA was isolated from LNCaP cells and from mixtures of LNCaP and MCF-7cells at fixed ratios (i.e. 1:100, 1:1000, etc.) using RNAzol B. NestedPCR was then performed as described below with both PSA and PSM primersin order to determine the limit of detection for the assay. LNCaP:MCF-7(1:100,000) cDNA was diluted with distilled water to obtainconcentrations of 1:1,000,000 and 1:10,000,000. MCF-7 cells were chosenbecause they have been previously tested and shown not to express PSM byPCR.

Polymerase Chain Reaction:

The PSA outer primers used span portions of exons 4 and 5 to yield a 486bp PCR product and ebabke differentiation between cDNA and possiblecontaminating genomic DNA amplification. The upstream primer sequencebeginning at nucleotide 494 in PSA cDNA sequence is5′-TACCCACTGCATCAGGAACA-3′ (SEQ ID NO: 38)and the downstream primer atnucleotide 960 is 5′-CCTTGAAGCACACCATTACA-3′ (SEQ ID NO: 39). The PSAinner upstream primer (beginning at nucleotide 559)5′-ACACAGGCCAGGTATTTCAG-3′ (SEQ ID NO: 40) and the downstream primer (atnucleotide 894) 5′ GTCCAGCGTCCAGCACACAG-3′ (SEQ ID NO:41) yield a 355 bpPCR product. All primers were synthesized by the MSKCC MicrochemistryCore Facility. 5μ—of total RNA was reverse-transcribed into cDNA in atotal volume of 20 μl using Superscript reverse transcriptase(Gibco-BRL) according to the manufacturers recommendations. 1 μl of thiscDNA served as the starting template for the outer primer PCR reaction.The 20 μl PCR mix included: 0.5U Taq polymerase (Promega Corp., Madison,Wis.), Promega reaction buffer, 1.5 mM MgCL₂. 200 mM dNTPs, and 1.0 μMof each primer. This mix was then transferred to a Perkin Elmer 9600 DNAthermal cycler and incubated for 25 cycles. The PCR profile was asfollows: 94 C×15 sec., 60 C×15 sec., and 72 C for 45 sec. After 25cycles, samples were placed on ice, and 1 μl of this reaction mix servedas the template for another round of PCR using the inner primers. Thefirst set of tubes were returned to the thermal cycler for 25 additionalcycles. PSM-PCR required the selection of primer pairs that also spannedan intron in order to be certain that cDNA and not genomic DNA werebeing amplified.

The PSM outer primers yield a 946 bp product and the inner primers a 434bp product. The PSM outer upstream primer used was5′-ATGGGTGTTTGGTGGTATTGACC-3′ (SEQ. ID. NO: 42) (beginning at nucleotide1404) and the downstream primer (at nucleotide 2348) was 5′TGCTTGGAGCATAGATGACATGC-3′ (SEQ ID NO: 43) The PSM inner upstream primer(at nucleotide 1581) was 5′-ACTCCTTCAAGAGCGTGGCG-3′ (SEQ. ID. NO: 44)and the downstream primer (at nucleotide 2015) was 5′AACACCATCCCTCCTCGAACC-3′ (SEQ. ID NO: 45). cDNA used was the same as forthe PSA assay. The 501 PCR mix included: 1U Taq Polymerase (Promega),250M dNTPs, 10 mM—mercaptoethanol, 2 mM MgCl₂, and 5 l of a 10× buffermix containing: 166 mM NH₄SO₄, 670 mM Tris pH 8.8, and 2 mg/ml ofacetylated BSA. PCR was carried out in a Perkin Elmer 480 DNA thermalcycler with the following parameters: 94 C×4 minutes for 1 cycle, 94C×30 sec., 58 C×1 minute, and 72 C×1 minute for 25 cycles, followed by72 C×10 minutes. Samples were then iced and 21 of this reaction mix wasused as the template for another 25 cycles with a new reaction mixcontaining the inner PSM primers. cDNA quality was verified byperforming control reactions using primers derived from—actin yielding a446 bp. PCR product. The upstream primer used was5′-AGGCCAACCGCGAGAAGATGA-3′ (SEQ. ID. NO: 46) (exon 3) and thedownstream primer was 5′-ATGTCACACTGGGGAAGC-3′ (SEQ ID NO: 47) (exon 4).The entire PSA mix and 10 l of each PSM reaction mix were run on 1.5-2%agarose gels, stained with ethidium bromide and photographed in an EagleEye Video Imaging System (Stratagene, Torrey Pines, Calif.) Assays wererepeated at least 3 times to verify results.

Cloning and Sequencing of PCR Products:

PCR products were cloned into the pCR II plasmid vector using the TAcloning system (Invitrogen). These plasmids were transformed intocompetent E. coli cells using standard methods (13) and plasmid DNA wasisolated using Magic Minipreps (Promega) and screened by restrictionanalysis. TA clones were then sequenced by the dideoxy method (14) usingSequenase (U.S. Biochemical). 3-4 g of each plasmid was denatured withNaOH and ethanol precipitated. Labeling reactions were carried outaccording to the manufacturers recommendations using ³⁵S-dATP (NEN), andthe reactions were terminated as discussed in the same protocol.Sequencing products were then analyzed on 6% polyacrilamide/7M urea gelsrun at 120 watts for 2 hours. Gels were fixed for 20 minutes in 10%methanol/10% acetic acid, transferred to Whatman 3MM paper and drieddown in a vacuum dryer for 2 hours at 80 C. Gels were thenautoradiographed at room temperature for 18 hours.

Southern Analysis:

Ethidium-stained agarose gels of PCR products were soaked for 15 minutesin 0.2N HCl, followed by 30 minutes each in 0.5N NaOH/1.5M NaCl and 0.1MTris pH 7.5/1.5M NaCl. Gels were then equilibrated for 10 minutes in10×SSC (1.5M NaCl/0.15M Sodium Citrate. DNA was transferred onto Nytrannylon membranes (Schleicher and Schuell) by pressure blotting in 10×SSCwith a Posi-blotter (Stratagene). DNA was cross-linked to the membraneusing a UV Stratalinker (Stratagene). Blots were pre-hybridized at 65 Cfor 2 hourthes and subsequently hybridized with denatured ³²P-labeled,random-primed cDNA probes (either PSM or PSA) (9,15). Blots were washedtwice in 1×SSPE/0.5% SDS at 42 C and twice in 0.1×SSPE/0.5% SDS at 50 Cfor 20 minutes each. Membranes were air-dried and autoradiographed for30 minutes to 1 hour at −70 C with Kodak X-Omat film.

Experimental Results

PCR amplification with nested primers improved the level of detection ofprostatic cells from approximately one prostatic cell per 10,000 MCF-7cells to better than one cell per million MCF-7 cells, using either PSAor PSM-derived primers (FIGS. 26 and 27). This represents a substantialimprovement in the ability to detect minimal disease. Characteristics ofthe 16 patients analyzed with respect to their clinical stage,treatment, serum PSA and PAP values, and results of the assay are shown.In total, PSA-PCR detected tumor cells in 2/30 patients (6.7%), whereasPSM-PCR detected cells in 19/30 patients (63.3%). There were no patientspositive for tumor cells by PSA and not by PSM, while PSM provided 8positive patients not detected by PSA. Patients 10 and 11 in table 1,both with very advanced hormone-refractory disease were detected by bothPSA and PSM. Both of these patients have died since the time thesesamples were obtained. Patients 4, 7, and 12, all of whom were treatedwith radical prostatectomies for clinically localized disease, and allof whom have non-measurable serum PSA values 1-2 years postoperativelywere positive for circulating prostatic tumor cells by PSM-PCR, butnegative by PSA-PCR. A representative ethidium stained gel photographfor PSM-PCR is shown in FIG. 28. Samples run in lane A represent PCRproducts generated from the outer primers and samples in lanes labeled Bare products of inner primer pairs. The corresponding PSM Southern blotautoradiograph is shown in FIG. 29. The sensitivity of the Southern blotanalysis exceeded that of ethidium staining, as can be seen in severalsamples where the outer product is not visible on FIG. 28, but isdetectable by Southern blotting as shown in FIG. 29. In addition, sample3 on FIGS. 28 and 29 (patient 6 in FIG. 30) appears to contain bothouter and inner bands that are smaller than the corresponding bands inthe other patients. DNA sequencing has confirmed that the nucleotidesequence of these bands matches that of PSM, with the exception of asmall deletion. This may represent either an artifact of PCR,alternative splicing of PSM mRNA in this patient, or a PSM mutation. Allsamples sequenced and analyzed by Southern analysis have been confirmedas true positives for PSA and PSM.

Experimental Details

The ability to accurately stage patients with prostate cancer at thetime of diagnosis is clearly of paramount importance in selectingappropriate therapy and in predicting long-term response to treatment,and potential cure. Pre-surgical staging presently consists of physicalexamination, serum PSA and PAP determinations, and numerous imagingmodalities including transrectal ultrasonography, CT scanning,radionuclide bone scans, and even MRI scanning. No present modality,however, addresses the issue of hematogenous micrometastatic disease andthe potential negative impact on prognosis that this may produce.Previous work has shown that only a fractional percentage of circulatingtumor cells will inevitably go on to form a solid metastasis (16),however, the detection of and potential quantification of circulatingtumor cell burden may prove valuable in more accurately staging disease.The long-term impact of hematogenous micrometastatic disease must bestudied by comparing the clinical courses of patients found to havethese cells in their circulation with patients of similar stage andtreatment who test negatively.

The significantly higher level of detection of tumor cells with PSM ascompared to PSA is not surprising to us, since more consistentexpression of PSM in prostate carcinomas of all stages and grades ascompared to variable expression of PSA in more poorly differentiated andanaplastic prostate cancers is noted. The detection of tumor cells inthe three patients that had undergone radical prostatectomies withsubsequent undetectable amounts of serum. PSA was suprising. Thesepatients would be considered to be surgical “cures” by standardcriteria, yet they apparently continue to harbor prostatic tumor cells.It will be interesting to follow the clinical course of these patientsas compared to others without PCR evidence of residual disease.

References of Example 3

-   1. Boring, C. C., Squires, T. S., and Tong, T.: Cancer    Statistics, 1993. CA Cancer J. Clin., 43:7-26, 1993.-   2. Lepor, H., and Walsh, P. C.: Long-term results of radical    prostatectomy in clinically localized prostate cancer: Experience at    the Johns Hopkins Hospital. NCI Monogr., 7:117-122, 1988.-   3. Bagshaw, M. A., Cox, R. S., and Ray, G. R.: Status of radiation    treatment of prostate cancer at Stanford University. NCI Monogr.,    7:47-60, 1988.-   4. Thompson, I. M., Rounder, J. B., Teague, J. L., et al.: Impact of    routine screening for adenocarcinoma of the prostate on stage    distribution. J. Urol., 137:424-426, 1987.-   5. Chiarodo, A.: A National Cancer Institute roundtable on prostate    cancer; future research directions. Cancer Res., 51:2498-2505, 1991.-   6. Wu, A., Ben-Ezra, J., and Colombero, A.: Detection of    micrometastasis in breast cancer by the polymerase chain reaction.    Lab. Invest., 62:109A, 1990.-   7. Fey, M. F., Kulozik, A. E., and Hansen-Hagge, T. E.: The    polymerase chain reaction: A new tool for the detection of minimal    residual disease in hematological malignancies. Eur. J. Cancer,    27:89-94, 1991.-   8. Moreno, J. G., Croce, C. M., Fischer, R., Monne, M., Vihko, P.,    Mulholland, S. G., and Gomella, L. G.: Detection of hematogenous    micrometastasis in patients with prostate cancer. Cancer Res.,    52:6110-6112, 1992.-   9. Israeli, R. S., Powell, C. T., Fair, W. R., and Heston, W. D. W.:    Molecular cloning of a complementary DNA encoding a    prostate-specific membrane antigen. Cancer Res., 53:227-230, 1993.-   10. Israeli, R. S., Powell, C. T., Corr, J. G., Fair, W. R., and    Heston, W. D. W.: Expression of the prostate-specific membrane    antigen (PSM).: Submitted to Cancer Research.-   11. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P.,    Rosenthal, H., Chu, T. M., Mirand, E. A., and Murphy, G. P.: LNCaP    model of human prostatic carcinoma. Cancer Res., 43:1809-1818, 1983.-   12. Soule, H. D., Vazquez, J., Long, A., Albert, S., and Brennan,    M.: A human cell line from a pleural effusion derived from a breast    carcinoma. J. Natl. Can. Inst., 51:1409-1416, 1973.-   13. Hanahan, D.: Studies on transformation of Escherichia coli with    plasmids. J. Mol. Biol., 166:557-580, 1983.-   14. Sanger, F., Nicklen, S., and Coulson, A. R.: DNA sequencing with    chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA,    74:5463-5467, 1977.-   15. Lundwall, A., and Lilja, H.: Molecular cloning of a human    prostate specific antigen cDNA. FEBS Letters, 214:317, 1987.-   16. Liotta, L. A., Kleinerman, J., and Saidel, G. M.: Quantitative    relationships of intravascular tumor cells, tumor vessels, and    pulmonary metastases following tumor implantation. Cancer Res.,    34:997-1003, 1974.

Example 4 Expression of the Prostate Specific Membrane Antigen (PSM)Diminishes the Mitogenic Stimulation of Aggressive Human ProstaticCarcinoma Cells by Transferrin

An association between transferrin and human prostate cancer has beensuggested by several investigators. It has been shown that the expressedprostatic secretions of patients with prostate cancer are enriched withrespect to their content of transferrin and that prostate cancer cellsare rich in transferrin receptors (J. Urol. 143, 381, 1990). Transferrinderived from bone marrow has been shown to selectively stimulate thegrowth of aggressive prostate cancer cells (PNAS 89, 6197, 1992). DNAsequence analysis has revealed that a portion of the coding region, fromnucleotide 1250 to 1700 possesses a 54% homology to the humantransferrin receptor. PC-3 cells do not express PSM mRNA or protein andexhibit increased cell growth in response to transferrin, whereas, LNCaPprostate cancer cells which highly express PSM have a very weak responseto transferrin. To determine whether PSM expression by prostatic cancercells impacts upon their mitogenic response to transferrin thefull-length PSM cDNA was transfected into the PC-3 prostate cancercells. Clones highly expressing PSM mRNA were identified by Northernanalysis and expression of PSM protein was verified by Western analysisusing the anti-PSM monoclonal antibody 7E11-C5.3.

2×10⁴ PC-3 or PSM-transfected PC-3 cells per well ere plated in RPMImedium supplemented with 10% fetal bovine serum and at 24 hrs. added 1μg per ml. of holotransferrin to the cells. Cells were counted at 1 dayto be highly mitogenic to the PC-3 cells. Cells were counted at 1 day todetermine plating efficiency and at 5 days to determine the effect ofthe transferrin. Experiments were repeated to verify the results.

PC-3 cells experienced an average increase of 275% over controls,whereas the LNCaP cells were only stimulated 43%. Growth kineticsrevealed that the PSM-transfected PC-3 cells grew 30% slower than nativePC-3 cells. This data suggests that PSM expression in aggressive,metastatic human prostate cancer cells significantly abrogates theirmitogenic response to transferrin.

The use of therapeutic vaccines consisting of cytokine-secreting tumorcell preparations for the treatment of established prostate cancer wasinvestigated in the Dunning R3327-MatLyLu rat prostatic adenocarcinomamodel. Only IL-2 secreting, irradiated tumor cell preparations werecapable of curing animals from subcutaneously established tumors, andengendered immunological memory that protected the animals from anothertumor challenge Immunotherapy was less effective when tumors wereinduced orthotopically, but nevertheless led to improved outcome,significantly delaying, and occasionally preventing recurrence of tumorsafter resection of the cancerous prostate. Induction of a potent immuneresponse in tumor bearing animals against the nonimmunogenic MatLyLutumor supports the view that active immunotherapy of prostate cancer mayhave therapeutic benefits.

Example 5 Cloning and Characterization of the Prostate Specific MembraneAntigen (PSM) Promoter

The expression and regulation of the PSM gene is complex. Byimmunostaining, PSM antigen was found to be expressed brilliantly inmetastasized tumor, and in organ confined tumor, less so in normalprostatic tissue and more heterogenous in BPH. PSM is strongly expressedin both anaplastic and hormone refractory tumors. PSM mRNA has beenshown to be down regulated by androgen. Expression of PSM RNA is alsomodulated by a host of cytokines and growth factors. Knowledge of theregulation of PSM expression should aid in such diagnostic andtherapeutic strategies as imunoscintigraphic imaging of prostate cancerand protate-specific promoter-driven gene therapy.

Sequencing of a 3 kb genomic DNA clone that contained 2.5 kb upstream ofthe transcription start site revealed that two stretches of about 300b.p. (−260 to −600; and −1325 to −1625) have substantial homology(79-87%) to known genes. The promoter lacks a GC rich region, nor doesit have a consensus TATA box. However, it contains a TA-rich region fromposition −35 to −65.

Several consensus recognition sites for general transcription factorssuch as AP1, AP2, NFkB, GRE and E2-RE were identified. Chimericconstructs containing fragments of the upstream region of the PSM genefused to a promoterless chloramphenicol acetyl transferase gene weretransfected into, and transiently expressed in LNCaP, PC-3, and SW620 (acolonic cell line). With an additional SV40 enhancer, sequence from −565to +76 exhibited promoter activity in LNCaP but not in PC-3 nor inSW620.

Materials and Methods

Cell Lines.

LNCaP and PC-3 prostatic carcinoma cell lines (American Type CultureCollection) were cultured in RPMI and MEM respectively, supplementedwith 5% fetal calf serum at 37° C. and 5% CO₂. SW620, a colonic cellline, is a gift from Melisa.

Polymerase Chain Reaction.

The reaction was performed in a 50 μl volume with a final concentrationof the following reagents: 16.6 mM NH₄SO₄, 67 mM Tris-HCl pH 8.8,acetylated BSA 0.2 mg/ml, 2 mM MgCl₂, 250 μM dNTPs, 10 mMβ-mercaptoethanol, and 1 U of rth 111 Taq polymerase (BoehringerMannhiem, Calif.). A total of 25 cycles were completed with thefollowing profile: cycle 1, 94° C. 4 min.; cycle 2 through 25, 94° C. 1min, 60° C. 1 min, 72° C. 1 min. The final reaction was extended for 10min at 72° C. Aliquots of the reaction were electrophoresed on 1%agarose gels in 1× Tris-acetate-EDTA buffer.

Cloning of PSM promoter.

A bacteriophage Pl library of human fibroblast genomic DNA (GenomicSystems, Inc., St. Lous., Mich.), was screened using a PCR method ofPierce et al. Primers located at the 5′ end of PSM cDNA were used:5′-CTCAAAAGGGGCCGGATTTCC-3′ (SEQ ID. NO: 48) and5′CTCTCAATCTCACTAATGCCTC-3′ (SEQ ID NO:49) A positive clone, P683, wasdigested with Xhol restriction enzyme. Southern analysis of therestricted fragments using a DNA probe from the extreme 5′ to the Ava-1site of PSM cDNA confirmed that a 3 Kb fragment contains the 5′regulatory sequence of the PSM gene. The 3 kb Xhol fragment wassubcloned into pKSBluescrpt vectors and sequenced using the dideoxymethod.

Functional Assay of PSM Promoter.

Chloramphenicol Acetyl Transferase, (CAT) gene plasmids were constructedfrom the Smal-HindIII fragments or subfragements (using eitherrestriction enzyme subfragments or PCR) by insertion into promoterlesspCAT basic or pCAT-enhancer vectors (Promega). pCAT-constructs werecotransfected with pSVβgal plasmid (5 μg of each plasmid) into celllines in duplicates, using a calcium phosphate method (Gibco-BRL,Gaithersburg, Md.). The transfected cells were harvested 72 hours laterand assayed (15 μg of lysate) for CAT activity using the LSC method andfor βgal activity (Promega). CAT activities were standardized bycomparision to that of the βgal activities.

Results

Sequence of the 5′ End of the PSM Gene.

The DNA sequence of the 3 kb XhoI fragment of p683 which includes. 500bp of DNA from the RNA start site was determined (FIGS. 31A-31D)Sequence 683XFRVS starts from the 5′ distal end of PSM promoter, itoverlaps with the published PSM putative promoter at nt 2485, i.e. theputative transcription start site is at nt 2485; sequence 683XF107 isthe reverse, complement of 683XFRVS). The sequence from the XhoIfragment displayed a remarkable arrays of elements and motifs which arecharacteristic of eukaryotic promoters and regulatory regions found inother genes (FIG. 32).

Functional Analysis of Upstream PSM Genomic Elements for PromoterActivity.

Various pCAT-PSM promoter constructs were tested for promoter activitiesin two prostatic cell lines: LNCaP, PC-3 and a colonic SW620 (FIG. 33).Induction of CAT activity was neither observed in p1070-CAT whichcontained a 1070 bp PSM 5′ promoter fragment, nor in p676-CAT whichcontained a 641 bp PSM 5′ promoter fragment. However, with an additionalSV-40 enhancer, sequence from −565 to +76 (p676-CATE) exhibited promoteractivity in LNCaP but not in PC-3 nor in SW620.

Therefore, a LNCaP specific promoter fragment from −565 to +76 has beenisolated which can be used in PSM promoter-driven gene therapy.

Example 6 Alternatively Spliced Variants of Prostate Specific MembraneAntigen RNA: Ratio of Expression as a Potential Measurement ofProgression

Materials and Methods

Cell Lines.

LNCaP and PC-3 prostatic carcinoma cell lines were cultured in RPMI andMEM respectively, supplemented with 5% fetal calf serum at 37° C. and 5%CO₂.

Primary tissues.

Primary prostatic tissues were obtained from MSKCC's in-house tumorprocurement service. Gross specimen were pathologically staged byMSKCC's pathology service.

RNA Isolation.

Total RNA was isolated by a modified guanidiniumthiocynate/phenol/chloroform method using a RNAzol B kit (Tel-Test,Friendswood, Tex.). RNA was stored in diethyl pyrocarbonate-treatedwater at −80° C. RNA was quantified using spectrophometric absorption at260 nm.

cDNA synthesis.

Two different batches of normal prostate mRNAs obtained from trauma-deadmales (Clontech, Palo Alto, Calif.) were denatured at 70° C. for 10min., then reverse transcribed into cDNA using random hexamers andSuperscript II reverse transcriptase (GIBCO-BRL, Gaithersburg, Md.) at50° C. for 30 min. followed by a 94° C. incubation for 5 min.

Polymerase Chain Reaction.

Oligonucleotide primers (5′-CTCAAAAGGGGCCGGATTTCC-3′(SEQ ID NO: 50) and5′-AGGCTACTTCACTCAAAG-3′)(SEQ ID NO: 51), specific for the 5′ and 3′ends of PSM cDNA were designed to span the cDNA sequence. The reactionwas performed in a 50 μl volume with a final concentration of thefollowing reagents: 16.6 mM NH₄SO₄, 67 mM Tris-HCl pH 8.8 m, acetylatedBSA 0.2 mg/ml. 2 mM MgCl₂, 250 μM dNTPs, 10 mM β-mercaptoethanol, and 1U of rTth polymerase (Perkin Elmer, Norwalk, Conn.). A total of 25cycles were completed with the following profile: cycle 1, 94° C. 4min.; cycle 2 through 25, 94° C. 1 min, 60° C. 1 min, 72° C. 1 min. Thefinal reaction was extended for 10 min at 72° C. Aliquots of thereaction were electrophoresed on 1% agarose gels in 1× Tris-acetate-EDTAbuffer.

Cloning of PCR Products.

PCR products were cloned by the TA cloning method into pCRII vectorusing a kit from Invitrogen (San Diego, Calif.). Ligation mixture weretransformed into competent Escherichia coli Inv5α.

Sequencing.

Sequencing was done by the dideoxy method using a sequenase kit from USBiochemical (Cleveland, Ohio). Sequencing products were electrophoresedon a 5% polyacrylamide/7M urea gel at 52° C.

RNase Protection Assays.

Full length PSM cDNA clone was digested with NgoM 1 and Nhe1. A 350 b.p.fragment was isolated and subcloned into pSPORT1 vector (GIBCO-BRL,Gaithersburg, Md.). The resultant plasmid, pSP350, was linearized, andthe insert was transcribed by SP6 RNA polymerase to yield antisenseprobe of 395 nucleotide long, of which 355 nucleotides and/or 210nucleotides should be protected from RNAse digestion by PSM or PSM′ RNArespectively (FIG. 2). Total celluar RNA (20 μg) from different tissueswere hybridized to the aforementioned antisense RNA probe. Assays wereperformed as described (7). tRNA was used as negative control. RPAs forLNCaP and PC-3 were repeated.

Results

RT-PCR of mRNA from Normal Prostatic Tissue.

Two independent RT-PCR of mRNA from normal prostates were performed asdescribed in Materials and Methods. Subsequent cloning and sequencing ofthe PCR products revealed the presence of an alternatively splicedvariant, PSM′. PSM′ has a shorter cDNA (2387 nucleotides) than PSM (2653nucleotides). The results of the sequence analysis are shown in FIG. 34.The cDNAs are identical except for a 266 nucleotide region near the 5′end of PSM cDNA (nucleotide 114 to 380) that is absent in PSM′ cDNA. Twoindependent repetitions of RT-PCR of different mRNA samples yieldedidentical results.

RNase Protection Assays.

An RNA probe complementary to PSM RNA and spanning the 3′ splicejunction of PSM′ RNA was used to measure relative expression of PSM andPSM′ mRNAs (FIG. 35). With this probe, both PSM and PSM′ RNAs in LNCaPcells was detected and the predominant form was PSM. Neither PSM norPSM′ RNA was detected in PC-3 cells, in agreement with previous Northernand Western blot data (5,6). FIG. 36 showed the presence of both splicevariants in human primary prostatic tissues. In primary prostatic tumor,PSM is the dominant form. In contrast, normal prostate expressed morePSM′ than PSM. BPH samples showed about equal expression of bothvariants.

Tumor Index.

The relative expression of PSM and PSM′ (FIG. 36) was quantified bydensitometry and expressed as a tumor index (FIG. 37). LNCaP has anindex ranging from 9-11; CaP from 3-6; BPH from 0.75 to 1.6; normalprostate has values from 0.075 to 0.45.

Discussion

Sequencing data of PCR products derived from human normal prostatic mRNAwith 5′ and 3′ end PSM oligonucleotide primers revealed a second splicevariant, PSM′, in addition to the previously described PSM cDNA.

PSM is a 750 a.a. protein with a calculated molecular weight of 84,330.PSM was hypothesized to be a type II integral membrane protein (5). Aclassic type II membrane protein is the transferrin receptor and indeedPSM has a region that has modest homology with the transferrin receptor(5). Analysis of the PSM amino acid sequence by either the methods ofRao and Argos (7) or Eisenburg et. al. (8) strongly predicted onetransmembrane helix in the region from a.a.#20 to #43. Both programsfound other regions that could be membrane associated but were notconsidered likely candidates for being transmembrane regions.

PSM′ antigen, on the other hand, is a 693 a.a. protein as deduced fromits mRNA sequence with a molecular weight of 78,000. PSM′ antigen lacksthe first 57 amino acids present in PSM antigen (FIG. 34). It is likelythat PSM′ antigen is cytosolic.

The function of PSM and PSM′ are probably different. The cellularlocation of PSM antigen suggests that it may interact with either extra-or intra-cellular ligand(s) or both; while that of PSM′ implies thatPSM′ can only react with cytosolic ligand(s). Furthermore, PSM antigenhas 3 potential phosphorylation sites on its cytosolic domain. Thesesites are absent in PSM′ antigen. On the other hand, PSM′ antigen has 25potential phosphorylation sites, 10 N-myristoylation sites and 9N-glycosylation sites. For PSM antigen, all of these potential siteswould be on the extracellular surface. The modifications of these sitesfor these homologous proteins would be different depending on theircellular locations. Consequently, the function(s) of each form woulddepend on how they are modified.

The relative differences in expression of PSM and PSM′ by RNaseprotection assays was analyzed. Results of expression of PSM and PSM′ inprimary prostatic tissues strongly suggested a relationship between therelative expression of these variants and the status of the cell: eithernormal or cancerous. While it is noted here that the sample size of thestudy is small (FIGS. 36 and 37), the consistency of the trend isevident. The samples used were gross specimens from patients. Theresults may have been even more dramatic if specimens that were pure incontent of CaP, BPH or normal had been used. Nevertheless, in thesespecimens, it is clear that there is a relative increase of PSM overPSM′ mRNA in the change from normal to CaP. The Tumor Index (FIG. 37)could be useful in measuring the pathologic state of a given sample. Itis also possible that the change in expression of PSM over PSM′ may be areason for tumor progression. A more differentiated tumor state may berestored by PSM′ either by transfection or by the use of differentiationagents.

References of Example 6

-   1. Murphy, G. P. Report on the American Urologic    Association/American Cancer Society Scientific Seminar on the    Detection and treatment of Early-Stage Prostate Cancer. CA Cancer J.    Clin. 44:91-95, 1994.-   2. Israeli, R. S., Miller Jr., W. H., Su, S. L., Powell, C. T.,    Fair,: W. R., Samadi, D. S., Huryk, R. F., DelBlasio, A.,    Edwards, E. T, and Heston, W. D. W. Sensitive Nested Reverse    Transcription Polymerase Chain. Reaction Detection of Circulating    Prostatic Tumor Cells: Comparision of Prostate-specific Membrane    Antigen and Prostate-specific Antigen-based Assays. Cancer Res., 54:    6325-6329,1994.-   3. Horoszewicz, J. S., Kawinski, E., and Murphy, G. P. Monoclonal    antibodies to a new antigenic marker in epithelial cells and serum    of prostatic cancer patients. Anticancer Res., 7:927-936, 1987.-   4. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P.,    Rosenthal, H., Chu, T. M., Mirand, E. A. and Murphy, G. P. LNCaP    model of human prostatic Carcinoma. Cancer Res., 43:1809-1818, 1983.-   5. Israeli, R. S., Powell, C. T., Fair, W. R. and Heston, W. D. W.    Molecular cloning of a complementary DNA encoding a    prostate-specific membrane antigen. Cancer Res., 53:227-230, 1993.-   6. Israeli, R. S., Powell, C. T., Corr, J. G., Fair, W. R. and    Heston, W. D. W. Expression of the prostate-specific membrane    antigen. Cancer Res., 54:1807-1811, 1994.-   7. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T.,    Zinn, K. and Green, M. R. Efficient in vitro synthesis of    biologically active RNA and RNA hybridization probes from plasmids    containing a bacteriophage SP6 promoter. Nucleic Acids Res.,    12:7035-7056, 1984.-   8. Rao, M. J. K. and Argos, P. A conformational preference parameter    to predict helices in integral membrane proteins. Biochim. Biophys.    Acta, 869:197-214, 1986.-   9. Eisenburg, D., Schwarz, E., Komaromy, M. and Wall, R. Analysis of    membrane and surface protein sequences with the hydrophbic moment    plot, J. Mol. Biol. 179:125-142, 1984.-   10. Troyer, J. K. and Wright Jr., G. L. Biochemical characterization    and mapping of 7E-11C-5.3. Epitope of the prostate specific membrane    antigen (PSMA). American Association for Cancer Research Special    Conference: Basic and Clinical Aspect of Prostate Cancer. Abstract    C-38, 1994.

Example 7 Enhanced Detection of Prostatic Hematogenous Micro-metastaseswith PSM Primers as Compared to PSA Primers Using a Sensitive NestedReverse Transcriptase-PCR Assay

77 randomly selected samples were analyzed from patients with prostatecancer and reveals that PSM and PSA primers detected circulatingprostate cells in 48 (62.3%) and 7 (9.1%) patients, respectively. Intreated stage D disease patients, PSM primers detected cells in 16 of 24(66.7%), while PSA primers detected cells in 6 of 24 patients (25%). Inhormone-refractory prostate cancer (stage D3), 6 of 7 patients werepositive with both PSA and PSM primers. All six of these patients diedwithin 2-6 months of their assay, despite aggressive cytotoxicchemotherapy, in contrast to the single patient that tested negativelyin this group and is alive 15 months after his assay, suggesting thatPSA-PCR positivity may serve as a predictor of early mortality. Inpost-radical prostatectomy patients with negative serum PSA values, PSMprimers detected metastases in 21 of 31 patients (67.7%), while PSAprimers detected cells in only 1 of 33 (3.0%), indicating thatmicrometastatic spread may be a relatively early event in prostatecancer. The analysis of 40 individuals without known prostate cancerprovides evidence that this assay is highly specific and suggests thatPSM expression may predict the development of cancer in patients withoutclinically apparent prostate cancer. Using PSM primers, micrometastaseswere detected in 4 of 40 controls, two of whom had known BPH by prostatebiopsy and were later found to have previously undetected prostatecancer following repeat prostate biopsy performed for a rising serum PSAvalue. These results show the clinical significance of detection ofhematogenous micrometastatic prostate cells using PSM primers andpotential applications of this molecular assay.

Example 8 Modulation of Prostate Specific Membrane Antigen (PSM)Expression IN VITRO by Cytokines and Growth Factors

The effectiveness of CYT-356 imaging is enhanced by manipulatingexpression of PSM. PSM mRNA expression is downregulated by steroids.This is consistent with the clinical observations that PSM is stronglyexpressed in both anaplastic and hormone refractory lesions. Incontrast, PSA expression is decreased following hormone withdrawal. Inhormone refractory disease, it is believed that tumor cells may produceboth growth factors and receptors, thus establishing an autocrine loopthat permits the cells to overcome normal growth constraints. Manyprostate tumor epithelial cells express both TGFα and its receptor,epidermal growth factor receptor. Results indicate that the effects ofTGFα and other selected growth factors and cytokines on the expressionof PSM in-vitro, in the human prostatic carcinoma cell line LNCaP.

2×10⁶ LNCaP cells growing in androgen-depleted media were treated for 24to 72 hours with EGF, TGFα, TNFβ or TNFα in concentrations ranging from0.1 ng/ml to 100 ng/ml. Total RNA was extracted from the cells and PSMmRNA expression was quantitated by Northern blot analysis and laserdensitometry. Both b-FGF and TGFα yielded a dose-dependent 10-foldupregulation of PSM expression, and EGF a 5-fold upregulation, comparedto untreated LNCaP. In contrast, other groups have shown a markeddownregulation in PSA expression induced by these growth factors in thissame in-vitro model. TNFα, which is cytotoxic to LNCaP cells, and TNFβdownregulated PSM expression 8-fold in androgen depleted LNCaP cells.

TGFα is mitogenic for aggressive prostate cancer cells. There aremultiple forms of PSM and only the membrane form is found in associationwith tumor progression. The ability to manipulate PSM expression bytreatment with cytokines and growth factors may enhance the efficacy ofCytogen 356 imaging, and therapeutic targeting of prostatic metastases.

Example 9 Neoadjuvant Androgen-deprivation Therapy (ADT) Prior toRadical Prostatectomy Results in a Significantly Decreased Incidence ofResidual Micrometastatic Disease as Detected by Nested RT-PCT withPrimers

Radical prostatectomy for clinically localized prostate cancer isconsidered by many the “gold standard” treatment. Advances over the pastdecade have served to decrease morbidity dramatically. Improvementsintended to assist clinicians in better staging patients preoperativelyhave been developed, however the incidence of extra-prostatic spreadstill exceeds 50%, as reported in numerous studies. A phase IIIprospective randomized clinical study designed to compare the effects ofADT for 3 months in patients undergoing radical prostatectomy withsimilarly matched controls receiving surgery alone was conducted. Thepreviously completed phase II study revealed a 10% margin positive ratein the ADT group (N=69) as compared to a 33% positive rate (N=72) in thesurgery alone group.

Patients who have completed the phase III study were analyzed todetermine if there are any differences between the two groups withrespect to residual micrometastatic disease. A positive PCR result in apost-prostatectomy patient identifies viable metastatic cells in thecirculation.

Nested RT-PCR was performed with PSM primers on 12 patients from the ADTgroup and on 10 patients from the control group. Micrometastatic cellswere detected in 9/10 patients (90%) in the control group, as comparedto only 2/12 (16.7%) in the ADT group. In the ADT group, 1 of 7 patientswith organ-confined disease tested positively, as compared to 3 of 3patients in the control group. In patients with extra-prostatic disease,1 of 5 were positive in the ADT group, as compared to 6 of 7 in thecontrol group. These results indicate that a significantly higher numberof patients may be rendered tumor-free, and potentially “cured” by theuse of neoadjuvant ADT.

Example 10 Sensitive Nested RT-PCR Detection of Circulation ProstaticTumor Cells—Comparison of PSM AND PSA-based Assays

Despite the improved and expanded arsenal of modalities available toclinician today, including sensitive serum PSA assays, CT scan,transrectal ultrasonography, endorectal co.I MRI, etc., many patientsare still found to have metastatic disease at the time of pelvic lymphnode dissection and radical prostatectomy. A highly sensitive reversetranscription PCR assay capable of detecting occult hematogenousmicrometastatic prostatic cells that would otherwise go undetected bypresently available staging modalities was developed. This assay is amodification of similar PCR assays performed in patients with prostatecancer and other malignancies^(2,3,4,5). The assay employs PCR primersderived from the cDNA sequences of prostate-specific antigen⁶ and theprostate-specific membrane antigen recently cloned and sequenced.

Materials and Methods

Cells and Reagents.

LNCaP and MCF-7 cells were obtained from the American Type CultureCollection (Rockville, Md.). Details regarding the establishment andcharacteristics of these cell lines have been previouslypublished^(8,9). Cells grown in RPMI 1640 medium and supplemented withL-glutamine, nonessential amino acids, and 5% fetal calf serum(Gibco-BRL, Gaithersburg, Md.) In a 5% CO₂ incubator at 37° C. All cellmedia was obtained from the MSKCC Media Preparation Facility. Routinechemical reagents were of the highest grade possible and were obtainedfrom Sigma Chemical Company (St. Louis, Mo.).

Patient Blood Specimens.

All blood specimens used in this study were from patients seen in theoutpatient offices of urologists on staff at MSKCC. Two anti-coagulatedtubes per patient were obtained at the time of their regularly scheduledblood draws. Specimens were obtained with informed consent of eachpatient as per a protocol approved by the MSKCC Institutional ReviewBoard. Samples were promptly brought to the laboratory for immediateprocessing. Seventy-seven specimens from patients with prostate cancerwere randomly selected and delivered to the laboratory “blinded” alongwith samples from negative controls for processing. These included 24patients with stage D disease (3 with D₀, 3 with D¹, 11 with D², and 7with D³), 31 patients who had previously undergone radical prostatectomyand had undetectable postoperative serum PSA levels (18 with pT2lesions, 11 with pT3, and 2 pT4), 2 patients with locally recurrentdisease following radical prostatectomy, 4 patients who had receivedeither external beam radiation therapy or interstitial I¹²⁵ implants, 10patients with untreated clinical stage T1-T2 disease, and 6 patientswith clinical stage T3 disease on anti-androgen therapy. The forty bloodspecimens used as negative controls were from 10 health males, 9 maleswith biopsy-proven BPH and elevated serum PSA levels, 7 healthy females,4 male patients with renal cell carcinoma, 2 patients with prostaticintraepithelial neoplasia (PIN), 2 patients with transitional cellcarcinoma of the bladder and a pathologically normal prostate, 1 patientwith acute prostatitis, 1 patient with acute promyelocytic leukemia, 1patient with testicular cancer, 1 female patient with renal cellcarcinoma, 1 patient with lung cancer, and 1 patient with a cyst of thetesticle.

Blood Sample Processing/RNA Extraction.

4 ml of whole anticoagulated venous blood was mixed with 3 ml of icecold PBS and then carefully layered atop 8 ml of Ficoll (Pharmacia,Uppsala, Sweden) in a 14-ml polystyrene tube. Tubes were centrifuged at200×g for 30 min. at 4° C. The buffy coat layer (approx. 1 ml.) wascarefully removed and rediluted to 50 ml with ice cold PBS in a 50 mlpolypropylene tube. This tube was then centrifuged at 2000×g for 30 min.at 4° C. The supernatant was carefully decanted and the pellet wasallowed to drip dry. One ml of RNazol B was then added to the pellet andtotal RNA was isolated as per manufacturers directions (Cinna/Biotecx,Houston, Tex.) RNA concentrations and purity were determined by UVspectroscopy on a Beckman DU 640 spectrophotometer and by gel analysis.

Determination of PCR Sensitivity.

RNA was isolated from LNCaP cells and from mixtures of LNCaP and MCF-7cells at fixed ratios (i.e. 1:100, 1:1,000, etc.) using RNAzol B. NestedPCR was then performed as described below with both PSA and PSM primersin order to determine the limit of detection for the assay. LNCaP:MCF-7(1:100,000) cDNA was diluted with distilled water to obtainconcentrations of 1:1,000,000. The human breast cancer cell line MCF-7was chosen because they had previously been tested by us and shown notto express either PSM nor PSA by both immunohistochemistry andconventional and nested PCR.

Polymerase Chain Reaction.

The PSA outer primer sequences are nucleotides 494-513 (sense) in exon 4and nucleotides 960-979 (anti-sense) in exon 5 of the PSA cDNA. Theseprimers yield a 486 bp PCR product from PSA cDNA that can bedistinguished from a product synthesized from possible contaminatinggenomic DNA.

PSA-494 5′-TAC CCA CTG CAT CAG GAA CA-3′ (SEQ ID NO: 38)

PSA-960 5′-CCT TGA AGC ACA CCA TTA CA-3′ (SEQ ID NO: 39)

The PSA inner upstream primer begins at nuclotide 559 and the downstreamprimer at nucleotide 894 to yield a 355 bp. PCR product.

PSA-559 5′-ACA CAG GCC AGG TAT TTC AG-3′ (SEQ ID NO: 40)

PSA-894 5′-GTC CAG CGT CCA-3′ (SEQ ID NO: 41)

All primers were synthesized by the MSKCC Microchemistry Core Facility.5μ of total RNA was reverse-transcribed into cDNA using random hexamerprimers (Gibco-BRL) according to the manufacturers recommendations. 1 μlof this CDNA served as the starting template for the outer primer PCRreaction. The 20 μl PCR mix included: 0.5U Taq polymerase (Promega)Promega reaction buffer, 1.5 mM MgCl₂, 200 μM dNTPs, and 1.0 μM of eachprimer. This mix was then transferred to a Perkin Elmer 9600 DNA thermalcycler and incubated for 25 cycles. The PCR profile was as follows: 94°C.×15 sec., 60° C.×15 sec., and 72° C. for 45 sec. After 25 cycles,samples were placed on ice, and 1 μl of this reaction mix served as thetemplate for another 25 cycles using the inner primers. The first set oftubes were returned to the thermal cycler for 25 additional cycles. ThePSM outer upstream primer sequences are nucleotides 1368-1390 and thedownstream primers are nucleotides 1995-2015, yielding a 67 bp PCRproduct.

PSM-1368 5′-CAG ATA TGT CAT TCT GGG AGG TC-3′ (SEQ ID. NO: 52)

PSM-2015 5′-AAC AAC ATC CCT CCT CGA ACC-3′ (SEQ ID. NO: 46)

The PSM inner upstream primer span nucleotides 1689-1713 and thedownstream primer span nucleotides 1899-1923, yielding a 234 bp PCRproduct.

PSM-1689 5′-CCT AAC AAA AGA GCT GAA AAG CCC-3′ (SEQ ID. NO: 53)

PSM-1923 5′-ACG GTG ATA CAG TGG ATA GCC GCT-3′ (SEQ ID. NO: 54)

2 μl of cDNA was used as the starting DNA template in the PCR assay. The50 μl PCR mix included: 1U Taq polymerase (Boehringer Mannheim), 250 μAMcNTPS, 10 mM β-mercaptoethanol, 2 mM MgCl₂, and 5 μl of a 10× buffer mixcontaining: 166 mM NH₄SO₄, 670 mM Tris pH 8.8 and 2 mg/ml of acetylatedBSA. PCR was carried out in a Perkin Elmer 480 DNA thermal cycler withthe following parameters: 94° C.×4 minutes for 1 cycle, 94° C.×30 sec.,58° C.×1 minute, and 72° C.×1 minute for 25 cycles, followed by 72°C.×10 minutes. Samples were then iced and 2.5 μl of this reaction mixwas used as the template for another 25 cycles with a new reaction mixcontaining the inner PSM primers. cDNA quality was verified byperforming control reactions using primers derived from the β-2microglobulin gene sequence¹⁰ a ubiquitous housekeeping gene. Theseprimers span exons 2-4 and generate a 620 bp PCR product. The sequencesfor these primers are:

β-2 (exon 2) 5′-AGC AGA GAA TGG AAA GTC AAA-3′ (SEQ ID NO:55)

β-2 (EXON 4) 5′-TGT TGA TGT TGG ATA AGA GAA-3′ (SEQ ID NO: 56)

The entire PSA mix and 7-10 μl of each PSM reaction mix ere run on1.5-2% agarose gels, stained with ethidium bromide and photographed inan Eage Eye Video Imaging System (Statagene, Torrey Pines, Calif.).Assays were repeated at least twice to verify results.

Cloning and Sequencing of PCR Products.

PCR products were cloned into the pCR II plasmid vector using the TAcloning system (Invitrogen). These plasmids were transformed intocompetent E. coli cells using standard methods¹¹ and plasmid DNA wasisolated using Magic Minipreps (Promega) and screened by restrictionanalysis. Double-stranded TA clones were then sequenced by the dideoxymethod¹² using ³⁵S-cCTP (NEN) and Sequenase (U.S. Biochemical).Sequencing products were then analyzed on 6% polyacrilamide/7M ureagels, which were fixed, dried, and autoradiographed as described.

Southern Analysis.

PCR products were transferred from ethidium-stained agarose gels toNytran nylon membranes (Schletcher and Schuell) by pressure blottingwith a Posi-blotter (stratagene) according to the manufacturer'sinstructions. DNA was cross-linked to the membrane using a UVStratalinker (Stratagene). Blots were pre-hybridized at 65° C. for 2hours and subsequently hybridized with denatured ³²P-labeled,random-primed¹³ cDNA probes (either PSA or PSM). Blots were washed twicein 1×SSC/0.5% SDS at 42° C. and twice in 0.1×SSC/0.1% SDS at 50° C. for20 minutes each. Membranes were air-dried and autoradiographed for 1-3hours at room temperature with Hyperfilm MP (Amersham).

Results

PSA and PSM Nested PCR Assays:

The application of nested PCR increased the level of detection from anaverage of 1:10,000 using outer primers alone, to better than1:1,000,000. Dilution curves demonstrating this added sensitivity areshown for PSA and PSM-PCR in FIGS. 1 and 2 respectively. FIG. 1 showsthat the 486 bp product of the PSA outer primer set is clearlydetectable with ethidium staining to 1:10,000 dilutions, whereas the PSAinner primer 355 bp product is clearly detectable in all dilutionsshown. In FIG. 2 the PSM outer primer 647 bp product is also clearlydetectable in dilutions to only 1:10,000 with conventional PCR, incontrast to the PSM inner nested PCR 234 bp product which is detected indilutions as low as 1:1,000,000. Southern blotting was performed on allcontrols and most of the patient samples in order to confirmspecificity. Southern blots of the respective dilution curves confirmedthe primer specificities but did not reveal any significantly increasedsensitivity.

PCR in Negative Controls:

Nested PSA and PSM PCR was performed on 40 samples from patients andvolunteers as described in the methods and materials section. FIG. 48reveals results from 4 representative negative control specimens, inaddition to a positive control. Each specimen in the study was alsoassayed with the β-2-microglobulin control, as shown in the figure, inorder to verify RNA integrity. Negative results were obtained on 39 ofthese samples using the PSA primers, however PSM nested PCR yielded 4positive results. Two of these “false positives” represented patientswith elevated serum PSA values and an enlarged prostate who underwent atransrectal prostate biopsy revealing stromal and fibromuscularhyperplasia. In both of these patients the serum PSA level continued torise and a repeat prostate biopsy performed at a later date revealedprostate cancer. One patient who presented to the clinic with atesticular cyst was noted to have a positive PSM nested PCR result whichhas been unable to explain. Unfortunately, this patient never returnedfor follow up, and thus have not been able to obtain another bloodsample to repeat this assay. Positive result were obtained with both PSAand PSM primers in a 61 year old male patient with renal cell carcinoma.This patient has a normal serum PSA level and a normal digital rectalexamination. Overall, if the two patients were excluded in whom apositive PCR, but no other clinical test, accurately predicted thepresence of prostate cancer, 36/38 (94.7%) of the negative controls werenegative with PSM primers, and 39/40 (97.5%) were negative using PSAprimers.

Patient Samples:

In a “blinded” fashion, in which the laboratory staff were unaware ofthe nature of each specimen, 117 samples from 77 patients mixed randomlywith 40 negative controls were assayed. The patient samples representeda diverse and heterogeneous group as described earlier. Severalrepresentative patient samples are displayed in FIG. 49, correspondingto positive results from patients with both localized and disseminateddisease. Patients 4 and 5, both with stage D prostate cancer exhibitpositive results with both the outer and inner primer pairs, indicatinga large circulating tumor cell burden, as compared to the other samples.Although the PSM and PSA primers yielded similar sensitivities in LNCaPdilution curves as previously shown, PSM primers detectedmicrometastases in 62.3% of the patient samples, whereas PSA primersonly detected 9.1%. In patients with documented metastatic prostatecancer (stages D₀-D₃) receiving anti-androgen treatment, PSM primersdetected micrometastases in 16/24 (66.7%), whereas PSA primers detectedcirculating cells in only 6/24 (25%). In the study 6/7 patients withhormone-refractory prostate cancer (stage D₃) were positive. In thestudy, PSA primers revealed micrometastatic cells in only 1/15 (6.7%)patients with either pT3 or pT4 (locally-advanced) prostate cancerfollowing radical prostatectomy. PSM primers detected circulating cellsin 9/15 (60%) of these patients. Interestingly, circulating cells 13/18(72.2%) patients with pT2 (organ-confined) prostate cancer followingradical prostatectomy using PSM primers was detected. None of thesepatient samples were positive by PSA-PCR.

Improved and more sensitive method for the detection of minimal, occultmicrometastic disease have been reported for a number of malignancies:by use of immunohistochemical methods (14), as well as the polymerasechain reaction (3, 4, 5). The application of PCR to detect occulthematogenous micrometastases in prostate cancer was first described byMoreno, et al. (2) using conventional PCR with PSA-derived primers.

When human prostate tumors and prostate cancer cells in-vitro werestudied by immunohistochemistry and mRNA analysis, PSM appeared to behighly expressed in anaplastic cells, hormone-refractory cells, and bonymetastases (22, 23, 24), in contrast to PSA. If cells capable ofhematogenous micrometastasis represent the more aggressive andpoorly-differentiated cells, they may express a higher level of PSM percell as compared to PSA, enhancing their detectibility by RT-PCR.

Nested RT-PCR assays are both sensitive and specific. Results have beenreliably reproduced on repeated occasions. Long term testing of bothcDNA and RNA stability is presently underway. Both assays are capable ofdetecting one prostatic cell in at least one million non-prostatic cellsof similar size. This confirms the validity of the comparison of PSM vs.PSA primers. Similar levels of PSM expression in both human prostaticcancer cells in-vivo and LNCaP cells in-vitro resulted. The specificityof the PSM-PCR assay was supported by the finding that two “negativecontrol” patients with positive PSM-PCR results were both subsequentlyfound to have prostate cancer. This suggests an exciting potentialapplication for this technique for use in cancer screening. In contrastto recently published data (18), significant ability for PSA primers toaccurately detect micrometastatic cells in patients with pathologicallywith pathologically organ-confined prostate cancer, despite thesensitivity of the assay failed to result. Rather a surprisingly highpercentage of patients with localized prostate cancer that harbor occultcirculating prostate cells following “curative” radical prostatectomyresults which suggests that micrometastasis is an early event inprostate cancer.

The application of this powerful new modality to potentially stageand/or follow the response to therapy in patients with prostate cancercertainly merits further investigation. In comparison to moleculardetection of occult tumor cells, present clinical modalities for thedetection of prostate cancer spread appear inadequate.

References for Example 10

-   1. Boring, C. C., Squires, T. S., Tong, T., and Montgomery, S.    Cancer Statistics, 1994. CA., 44: 7-26, 1994.-   2. Moreno, J. G., Croce, C. M., Fischer, R., Monne, M., Vihko, P.,    Mulholland, S. G., and Gomella, L. G., Detection of hematogenous    micrometastasis in patients with prostate cancer. Cancer Res.,    52:6110-6112, 1992.-   3. Wu, A., Ben-Ezra, J., and Colombero, A.: Detection of    micrometastasis in breast-cancer by the polymerase chain reaction.    Lab. Ivest., 62: 109A, 1990.-   4. Fey, M. F., Kulozik, A. E., and Hansen-Hagge, T. E.: The    polymerase chain reactipn: A new tool for the detection of minimal    residual disease in hematological malignacies. Eur. J. Cancer, 27:    89-94, 1991.-   5. Miller, W. H., Jr., Levine, K., DeBlasio, A., Frankel, S. R.,    Dmitrovsky, E., and Warrell, R. P., Jr. Detection of mininal    residual disease in Acute Promyelocytic Leukemia by a reverse    transciption polymerase chain reaction assay for th PML/RAR-α fusion    mRNA. Blood, 82: 1689-1694, 1993.-   6. Lundwall, A., and Lilja, H: Molecular cloning of a human prostate    specific antigen cDNA. FEBS Letters, 214: 317, 1987.-   7. Isaeli, R. S., Powell, C. T., Fair, W. R., and Heston, W. D. W.:    Molecular cloning of a complementary DNA encoding a    prostate-specific membran antigen. Cancer Res., 53: 227-230, 1993.-   8. Horoszewicz, J. S., Leong, S. S., Kawinski, E., Karr, J. P.,    Rosenthal, H., Chu, T. M., Mirand, E. A., and Murphy, G. P.: LNCaP    model of human prostactic carcinoma. Cancer Res., 43: 1809-1818,    1983.-   9. Soule, H. D., Vazquez, J., Long, A., Albert, S., and Brennan, M.:    A human cell line from a pleural effusion derived from a breast    carcinoma. J. Natl. Can. Inst., 51: 1409-1416, 1973.-   10. Gussow, D., Rein, R., Ginjaar, I., Hochstenbach, F., Seemann,    G., Kottman, A., Ploegh, H. L. The human β-2-Microglobulin gene.    Primary structure and definition of the transcriptional unit. J. of    Immunol. 139:3132-3138, 1987.-   11. Hanahan, D.: Studies on transformation of Escherichia coli with    plasmids. J. Mol. Biol., 166:557-580, 1983.-   12. Sanger, F., Nicklen, S., and Coulson, A. R.: DNA sequncing with    chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA,    74:5463-5467, 1977.-   13. Feinberg, A. P., and Vogelstein, B. A technique for    radiolabeling DNA restriction endonuclease fragments to high    specific activity. Anal. Biochem., 132:6-13, 1983.-   14. Oberneder, R., Riesenberg, R., Kriegmair, M., Bitzer, U.,    Klammert, R., Schneede, P., Hofstetter, A., Riethmuller, G., and    Pantel, K. Immunocytochemcical detection and phenytypic    characterization of micrometastatic tumour cells in bone marrow of    patients with prostate cancer. Urol. Res. 22:3-8, 1994.

15. Israeli, R. S., Miller, W. H., Jr., Su, S. L., Samadi, D. S.,Powell, C. T., Heston, W. D. W., Wise, G. J., and Fair, W. R. Sensitivedetection of prostatic hematogenous micrometastases usingprostate-specific antigen (PSA) and prostate-specific membran antigen(PSM) derived primers in the polymerase chain reaction. J. Urol.151:373A, 1994.

-   16. Israeli, R. S., Miller, W. H., Jr., Su, S. L, Samadi, D. S.,    Powell, C. T. Heston, W. D. W., Wise, G. J., and Fair, W. S.    Sensitive detection of prostatic hematogenous micrometastases using    PsA and PSM-derived primers in the polymerase chain reaction. In    press—J. Urology.-   17. Vessella, R., Stray, J., Arman, E., Ellis, W., and Lange, P.    Reverse transcription polymerase chain reaction (RT-PCR) detects    metastatic prostate cancer cells in lymph nodes, blood and    potentially bone marrow using PSA-mRNA as template, J. Urol.    151:412A, 1994.-   18. Katz, A. E., Olsson, C. A., Raffo, A. J., Cama, C., Perlman, H.,    Seaman, E., O'Toole, K. M., McMahon, D., Benson, M., and Buttyan,    R., Molecular staging of prostate cancer with the use of an enhanced    reverse transcriptase-PCR assay. Urology 43:765-775, 1994.-   19. Wood, D. P., Jr., Banks, E. R., Humphries, S., McRoberts, J. W.,    and Rangenkar, V. M. Identification of micrometastases in paitents    with prostate cancer. J. Urol. 151:303A, 1994.-   20. Deguchi, T., Doi, T., Ehara, H., Ito, S., Takahashi, Y.,    Nishino, Y., Fujihiro, S., Kawamura, T., Komeda, H., Horie, M.,    Kaji, H., Shimokawa, K., Tanaka, T., and Kawada, Y. Detection of    micrometastic prostate cancer cells in lymph nodes by    reverse-transcriptase polymerase chain reaction. Cancer Res.    53:5350-4, 1993.-   21. Ghossein, R., Scher, H., Gerald, W., Hoffman, A., Kelley, W.,    Curely, T., Libertz, C., and Rosai, J. Detection of cirulating tumor    cells in peripheral blood of patients with advanced prostatic    carcinoma. Proc. Amer. Soc. of Clin. Oncol., 13:237, 1994.-   22. Israeli, R. S., Powel, C. T., Corr, J. G., Fair, W. R., and    Heston, W. D. W.: Expression of the prostate-specific membrane    antigen. Cancer Res., 54:1807-1811, 1994.-   23. Axelrod, H. R., Gilman, S. C., D'Aleo, C. H. Petrylak, D.,    Reuter, V., Gulfo, J. V., Saad A., Cordon-Cardo, C., and    Scher, H. I. Preclinical results and human immunohistochemical    strudies with ⁹⁰Y-CYT-356: a new prostatic cancer therapeutic    agent. J. Urol., 147:361A, 1992.-   24. Wright, G. L., Jr., Haley, C., Beckett, M. L., and    Schellhammer, P. F. Expression of the prostate biomaker 7E11-C5 in    primary and metastic prostate carcinoma. Proc. Amer. Ass. for Can.    Res. 35:233, 1994.-   25. Liotta, L. A., Kleinerman, J., and Saidel, G. M.: Quantitative    relationships of intravascular tumor cells, tumors vessels, and    pulmonary metastases following tumore implantation. Cancer Res.,    34:997-1003, 1974.

Example 11 Chromosomal Localization of Cosmid Clones 194 and 683 byFluorescence IN-SITU Hybridization

PSM was initially mapped as being located on chromosome 11p11.2-p13(FIGS. 51-54). Further information from the cDNA in-situ hybridizationsexperiments demonstrated as much hybridization on the q as p arms. Muchlarger fragments of genomic DNA was obtained as cosmids and two of theseof about 60 kilobases each one going 3′ and the other 5′ bothdemonstrated binding to chromosome 11 p and q under low stringency.However under higher stringency conditions only the binding at 11q14-q21remained. This result suggests that there is another gene on 11p that isvery similar to PSM because it is so strongly binding to nearly 120kilobases of genomic DNA (FIG. 50).

Purified DNA from cosmid clones 194 and 683 was labelled with biotindUTP by nick translation. Labelled probes were combined with shearedhuman DNA and independently hybridized to normal metaphase chromosomesderived from PHA stimulated peripheral blood lymphocytes in a solutioncontaining 50% formamide, 10% dectran sulfate, and 2×SSC. Specifichybridization signals were detected by incubating the hybridized slidesin fluoresein conjugated avidin. Following signal detection the slideswere counterstained with propidium iodide and analyzed. These firstexperiments resulted in the specific labelling of a group C chromosomeon both the long and short arms. This chromosome was believed to bechromosome 11 on the basis of its size and morphology. A second set ofexperiments were performed in which a chromosome 11 centromere specificprobe was cohybridized with the cosmid clones. These experiments werecarried out in 60% formamide in an attempt to eliminate the crossreactive signal which was observed when low stringency hybridizationswere done. These experiments resulted in the specific labelling of thecentromere and the long arm of chromosome 11. Measurements of 10specifically labelled chromosomes 11 demonstrated that the cosmid clonesare located at a position which is 44% of the distance from thecentromere to the telomere of chromosome arm 11q, an area thatcorresponds to band 14q. A total of 160 metaphase cells were examinedwith 153 cells exhibiting specific labelling.

Cloning of the 5′ upstream and 3′ downstream regions of the PSM genomicDNA. A bacteriophage P1 library of human fibroblast genomic DNA (GenomicSystems, St. Louis, Mich.) was screened using the PCR method of Pierceet. al. Primer pairs located at either the 5′ or 3′ termini of PSM cDNAwere used. Positive cosmid clones were digested with restriction enzymesand confirmed by Southern analysis using probes which were constructedfrom either the 5′ or 3′ ends of PSM cDNA. Positive clone p683 containsthe 5′ region of PSM cDNA and about 60 kb upstream region. Clone-194contains the 3′ terminal of the PSM cDNA and about 60 kb downstream.

Example 12 Peptidase Enzymatic Activity

PSM is a type two membrane protein. Most type two membrane proteins arebinding proteins, transport proteins or peptidases. PSM appears to havepeptidase activity. When examining LNCaP cells with a substrateN-acetyl-aspartyl-¹⁴C-glutamic acid, NAAG, glutamic acid was released,thus acting as a carboxypeptidase. In vitro translated PSM message alsohad this peptidase activity.

The result is that seminal plasma is rich in its content of glutamicacid, and are able to design inhibitors to enhance the activity of thenon degraded normal substrate if its increased level will have abiologic desired activity. Also biologic activity can be measured to seehow it correlates wit the level of message. Tissue may be examined foractivity directly rather than indirectly using in-situ analysis orimmunohistochemical probes. Because there is another gene highly similaron the other arm of chromosome 11 when isolated the expressed clonedgenes can be used to determine what are the substrate differences anduse those substrates for identification of PSM related activity, say incirculating cells when looking for metastases.

Example 13 Ionotropicglutamate Receptor Distribution in Prostate Tissue

Introduction:

Excitatory neurotransmission in the central nervous system (CNS) ismediated predominantly by glutamate receptors. Two types of glutamatereceptors have been identified in human CNS: metabotropic receptors,which are coupled to second-messenger systems, and ionotropic receptors,which serve as ligand-gated ion channels. The presence of ionotropicglutamate receptors in human prostate tissue was investigated.

Methods:

Detection of glutamate receptor expression was performed usinganti-GluR2/3 and anti-biotin immunohistochemical technique inparaffin-embedded human prostate tissues. PSM antigen is aneurocarboxypeptidase that acts to release glutamate. In the CNSglutamate acts as a neurotransmitter by acting on glutaminergic ionchannels and increases the flow of ions like calcium ions. One way theglutamate signal is transduced into cell activity is the activation ofnitric oxide synthase, and nitric oxide synthase has recently been foundto be present in human prostatic tissue. NO is a major signallingmechanism and is involved in control of cell growth and death, inresponse to inflammation, in smooth muscle cell contraction, etc. In theprostate much of the stroma is smooth muscle. It was discovered that theprostate is rich in glutaminergic receptors and have begun to definethis relationship. Stromal abnormalities are the key feature of BPH.Stromal epithelial interactions are of importance in bothe BPH and CaP.The other glutaminergic receptors through G proteins to change themetabolism of the cell.

Results

Anti-GluR2/3 immunoreactivity was unique to prostatic stroma and wasabsent in the prostatic epithelial compartment. Strong anti-GluR4immunoreactivity was observed in basal cells of prostatic acini.

Discussion:

The differential distribution of ionotropic glutamate receptor subtypesbetween the stromal and epithelial compartments of the prostate has notbeen previously described. Prostate-specific membrane antigen (PSMA) hasan analogous prostatic distribution, with expression restricted to theepithelial compartment.

PSM antigen is a neurocarboxypeptidase that acts to release glutamatefrom NAAG 1, also a potential nerotransmitter. In the CNS glutamate actsas a neurotransmitter by acting on glutaminergic ion channels andincreases the flow of ions like calcium ions. One way the glutamatesignal is transduced into cell activity is the activation of nitricoxide synthase, and nitric oxide synthase has recently been found to bepresent in human prostatic tissue. NO is a major signaling mechanism andis involved in control of cell growth and death, in response toinflammation, in smooth muscle cell contraction, etc,. In the prostatemuch of the stroma is smooth muscle. The prostate is rich inglutaminergic receptors. Stromal abnormalities are the key feature ofBPH. Stromal epithelial interactions are of importance in both BPH andCaP. The other glutaminergic receptors through G proteins to change themetabolism of the cell. Glutamate can be produced in the cerebral cortexthrough the carboxypeptidase activity of the prostate-specific membraneantigen (PSMA). In this location, PSMA cleaves glutamate fromacetyl-aspartyl-glutamate. Taken together, these observations suggest afunction for PSMA in the human prostate; glutamate may be an autocrineand/or paracrine signalling molecule, possibly mediatingepithelial-stromal interactions. Ionotropic glutamate receptors displaya unique compartmental distribution in the human prostate.

The carboxypeptidase like activity and one substrate is the dipeptideN-acetyl-aspartyl glutamic acid, NAAG which is one of the bestsubstrates found to date to act as a neurotransmitter in the centralnervous system and its abnormal function may be associated withneurotoxic disorder such as epilepsy, ALS, alzheimers etc. PSMcarboxypeptidase may serve to process neuropeptide transmitters in theprostate. Neuropeptide transmitters are associated with theneuroendocrine cells of the prostate and neuroendocrine cells and arethought to play a role in prostatic tumor progression. Interestingly PSMantigen's expression is upregulated in cancer. Peptides known to act asprostatic growth factors such as TGF-a and bFGF, up regulate theexpression of the antigen. TNF on the other hand downregulate PSM. TGFand FGF act through the mitogen activated signaling pathway, while TNFacts through the stress activated protein kinase pathway. Thusmodulation of PSM expression is useful for enhancing therapy.

Example 14 Identification of a Membrane-bound Pteroylpolygamma-glutamylCarboxypeptidase (Folate Hydrolase) that is Expressed in Human ProstaticCarcinoma

PSM may have activities both as a folate hydrolase and acarboxyneuropeptidase. For the cytotoxic drug methotrexate to be a tumortoxin it has to get into the cell and be polygammaglutamated which to beactive, because polyglutamated forms serve as the enzyme substrates andbecause polyglutamated forms or toxins are also retained by the cell.Folate hydrolase is a competing reaction and deglutamates methotrexatewhich then can diffuse back out of the cell. Cells that overexposefolate hydrolase activity are resistant to methotrexate. Prostate cancerhas always been absolutely refractory to methotrexate therapy and thismay explain why, since the prostate and prostate cancer has a lot offolate hydolase activity. However, based on this activity, prodrugs maybe generated which would be activate at the site of the tumor such asN-phosphonoacetyl-1-aspartate-glutamate. PALglu is an inhibitor of theenzyme activity with NAAG as a substrate.

Prostate specific membrane antigen was immuno precipitated from theprostate cancer cell line LNCaP and demonstrated it to be rich in folatehydolase activity, with gammaglutamated folate or polyglutamatedmethotrexate being much more potent inhibitors of the neuropeptidaseactivity than was quisqualate, which was the most potent inhibitorreported up to this time and consistent with the notion thatpolyglutamated folates may be the preferred substrate.

Penta-gammaglutamyl-folate is a very potent inhibitor of activity(inhibition of the activity of the enzyme is with 0.5 um Ki.) Aspenta-gammaglutamyl-folate may also be a substrate and as folates haveto be depolygammaglutamated in order to be transported into the cell,this suggest that this enzyme may also play a role in folate metabolism.Folate is necessary for the support of cell function and growth and thusthis enzyme may serve to modulate folate access to the prostate andprostate tumor. The other area where PSM is expressed is in the smallintestine. It turns out that a key enzyme of the small intestine that isinvolved in folate uptake acts as a gamma-carboxypeptidase insequentially proteolytically removing the terminal gammaglutaminyl groupfrom folate. In the bone there is a high level of unusual gammaglutamatemodified proteins in which the gamma glutamyl group is furthercarboxylated to produce gammacarboxyglutamate, or GLA. One such proteinis osteonectin.

Using capillary electrophoresisis pteroyl poly-gamma-glutamatecarboxypeptidase (hydrolase) activity was investigated in membranepreparations from androgen-sensitive human prostatic carcinoma cells(LNCaP). The enzyme immunologically cross-reacts with a derivative of ananti-prostate monoclonal antibody (7E11-C5) that recognizes prostatespecific membrane (PSM) antigen. The PSM enzyme hydrolyzesgamma-glutamyl linkages and is an exopeptidase as it liberatesprogressively glutamates from methotrexate triuglutamate (MTXGlu₃) andfolate pentaglutamate (Pte Glu₃) with accumulation of MTX and Pte Glurespectively. The semi-purified membrane-bound enzyme has a broadactivity from pH 2 to 10 and is maximally active at pH4.0. Enzymaticactivity was weakly inhibited by dithfothreitol (≧0.2 mM) but not byreduced glutathione, homocysteine, or p-hydroxymercuribenzoate (0.05-0.5mM). By contrast to LNCaP cell membranes, membranes isolated fromandrogen-insensitive human prostate (TSU-Prl, Duke-145, PC-3) andestrogen-sensitive mammary adenocarcinoma (MCF-7) cells do not exhibitcomparable hydrolase activity nor do they react with 7E11-C5. Thus, afolate hydrolase was identified in LNCap cells that exhibitsexopeptidase activity and is strongly expressed by these cells.

PALA-Glutamate 3 was tested for efficacy of the prodrug strategy bypreparing N-acetylaspartylglutamate, NAAG 1 (FIG. 59). NAAG wassynthesized from commercially available gamma-benzylaspartate which wasacetylated with acetic anhydride in pyridine to affordN-acetyl-gamma-benzyl aspartate in nearly quantitative yield. The latterwas activated as its pentafluorophenyl ester by treatment withpentafluorophenyltrifluoroacetate in pyridine at 0 deg. C. for an hour.This activated ester constitutes the central piece in the preparation ofcompounds 1 and 4 (FIG. 60). When 6 is reacted withepsilon-benzyl-L-glutamate in the presence ofHOAT(1-hydroxy-7-azabenzotriazole) in THF-DMF (tetrahydrofuran,N,N-dimethylformamide) at reflux for an overnight period and afterremoval of the benzyl protecting groups by hydrogenolysis (H2, 30 psi,10% Pd/C in ethylacetate) gave a product which was identical in allrespects to commercially available NAAG (sigma).

PALA-Glutamate 3 and analog 5, was synthesized in a similar manner withthe addition to the introduction of a protected phosphonoacetate moietyinstead of a simple acetate. It is compatible with the function ofdiethylphosphonoacetic acid which allows the removal of the ethyl groupsunder relatively mild conditions.

Commercially available diethylphosphonoacetic acid was treated withperfluorophenyl acetate in pyridine at 0 deg. C. to room temperature foran hour to afford the corresponding pentafluorophenyl ester in nearlyquantitative yield after short path column chromatography. This was thenreacted with gamma-benzylaspartate and HOAT in tetrahydrofuran for halfan hour at reflux temperature to give protected PALA 7(N-phosphonoacetylaspartate) in 90% yield after flash columnchromatography. The free acid was then activated as itspentafluorophenyl ester 8, then it was reacted withdelta-benzyl-L-glutamate and HOAT in a mixture of THF-DMF (9:1, v/v) for12 hours at reflux to give fully protected PALA-Glutamate 9 in 66% yieldafter column chromatography. Sequential removal of the ethyl groupsfollowed by the debenzylation was accomplished for a one stepdeprotection of both the benzyl and ethyl groups. Hence protectedPALA-Glutamate was heated up to reflux in neat trimethylsilylchloridefor an overnight period. The resulting bistrimethylsilylphosphonateester 10 was submitted without purification to hydrogenolysis (H₂,30psi, 10% Pd/C, ethylacetate). The desired material 3 was isolated afterpurification by reverse phase column chromatography and ion exchangeresin.

Analogs 4 and 5 were synthesized by preparation of phosphonoglutamate 14from the alpha-carboxyl-protected glutamate.

Commercially available alpha-benzyl-N-Boc-L-glutamate 11 was treated atrefluxing THF with neat boranedimethylsulfide complex to afford thecorresponding alcohol in 90% yield. This was transformed into bromide 12by the usual procedure (Pph₃,CBr₄).

The Michaelis-Arbuzov reaction using triethylphosphite to give thecorresponding diethylphosphonate 13 which would be deprotected at thenitrogen with trifluoroacetic acid to give free amine 14. The latterwould be condensed separately with either pentafluorophenylesters 6 or 8to give 16 and 15 respectively, under conditions similar to thosedescribed for 3. 15 and 16 would be deprotected in the same manner asfor 3 to yield desired analogs 4 and 5.

An inhibitor of the metabolism of purines and pyrimidine like DON(6-diazo-5-oxo-norleucine) or its aspartate-like 17, and glutamate-like18 analogs would be added to the series of substrates.

Analog 20 is transformed into compound 17 by treatment with oxalylchloride followed by diazomethane and deprotection under knownconditions to afford the desired analogs. In addition, azotomycin isactive only after in vivo conversion to DON which will be released afteraction of PSM on analogs 17, 18, and 19.

In addition, most if not all chemotherapies rely on one hypothesis; fastgrowing cells possess a far higher appetite for nutrients than normalcells. Hence, they uptake most of the chemotherapeutic drugs in theirproximity. This is why chemotherapy is associated with serious secondaryeffects (weakening of the immune system, loss of hair, . . . ) thatsometimes put the patient's life in danger. A selective and effectivedrug that cures where it should without damaging what it shouldn'tdamage is embodied in representative structures 21 and 22.

Representative compounds, 21 and 22, were designed based on some of thespecific effects and properties of PSM, and the unique features of somenewly discovered cytotoxic molecules with now known mode of action. Thelatter, referred to commonly as enediynes, like dynemycin A 23 and orits active analogs. The recent isolation of new natural products likeDynemycin A 23, has generated a tremendous and rapidly growing interestin the medical and chemical sciences. They have displayed cytotoxicitiesto many cancer cell lines at the sub-nanomolar level. One problem isthey are very toxic, unstable, and non-selective. Although they havebeen demonstrated, in vitro, to exert their activity through DNA damageby a radical mechanism as described below, their high level of toxicitymight imply that they should be able to equally damage anything in theirpath, from proteins to enzymes, . . . etc.

These molecules possess unusual structural features that provide themwith exceptional reactivities. Dynemycin A 23 is relatively stable untilthe anthraquinone moiety is bioreduced into hydroanthraquinone 24. Thistriggers a chain of events by which a diradical species 25 is generatedas a result of a Bergman cycloaromatization^(F). Diradical species 25 isthe ultimate damaging edge of dynemycin A. It subtracts 2 (two) protonsfrom any neighboring molecule or molecules (ie. DNA) producing radicalstherein. These radicals in turn combine with molecular oxygen to givehydroperoxide intermediates that, in the case of DNA, lead to single anddouble strand incision, and consequent cell death. Another interestingfeature was provided by the extensive work of many organic chemists whonot only achieved the-total synthesis of (+)-dynemycin A 23 and otherenediynes. but also designed and efficiently prepared simpler yet asactive analogs like 26.

Enediyne 26 is also triggerable and acts by virtue of the same mechanismas for 23. This aspect is very relevant to the present proposed study inthat 27 (a very close analog of 26) is connected to NAAG such that theNAAG-27 molecule, 21, would be inert anywhere in the body (blood,organs, normal prostate cells, . . . etc.) except in the vicinity ofprostate cancer, and metastatic cells. In this connection NAAG plays amultiple role:

-   -   Solubilization and transport: analogs of 26-type are hydrophobic        and insoluble in aqueous media, but with a water soluble        dipeptide that is indigenous to the body, substrate 21 should        follow the ways by which NAAG is transported and stored in the        body.    -   Recognition, guidance, and selectivity: Homologs of PSM are        located in the small intestines and in the brain.

In the latter, a compound like 27 when attached to a multiply chargeddipeptide like NAAG, has no chance of crossing the blood brain barrier.In the former case, PSM homolog concentration in the small intestines isvery low compared to that of PSM in prostrate cancer cells. In addition,one could enhance the selectivity of delivery of the prodrug by localinjection in the prostate. Another image of this strategy could beformulated as follows. If prostate cancer were a war in which one neededa “smart bomb” to minimize the damage within the peaceful surroundingsof the war zone, then 21 would be that “smart bomb”. NAAG would be itsguidance system, PSM would be the trigger, and 27 would be the warhead.

26 and its analogs are established active molecules that portray theactivity of dynemycin A. Their syntheses are described in theliterature. The total synthesis of optically active 27 has beendescribed. The synthetic scheme that for the preparation of 28 is almostthe same as that of 27. However, they differ only at the position of themethoxy group which is meta to the nitrogen in the case of 28. Thisrequires an intermediate of type 29, and this is going to be prepared bymodification of the Myers' method. Compound 28 is perhaps the closestoptically active analog that resembles very much 26, and since theactivity of the latter is known and very high.

Since NAAG is optically pure, its combination with racemic materialsometimes complicates purification of intermediates. In addition, to beable to modify the components of this system one at a time, opticallypure intermediates of the type 21 and 22 are prepared. 27 was preparedin 17 steps starting fro commercially available material. Anotherinteresting feature of 27 is as demonstrates in a very close analog 26,it possesses two(2) triggers as shown by the arrows.

The oxygen and the nitrogen can both engender the Bergmancycloaromatization and hence the desired damage. The simple protectiondeprotection manipulation of either functionality should permit theselective positioning of NAAG at the nitrogen or at the oxygen centers.PSM should recognize the NAAG portion of 21 or 22, then it would removethe glutamic acid moiety. This leaves 27 attached to N-acetylaspartate.

Intramolecular assisted hydrolysis of systems like N-acetylaspartyle iswell documented in the literature. The aminoacid portion shouldfacilitate the hydrolysis of such a linkage. In the event this would notwork when NAAG is placed on the nitrogen, an alternative would be toattach NAAG to the oxygen giving rise to phenolic ester 22 which is perse labile and removable under milder conditions. PSM specific substratescan be designed that could activate pro-drugs at the site of prostatictumor cells to kill those cells. PSM specific substrates may be used intreatment of benign prostatic hyperplasia.

Example 15

GENOMIC ORGANIZATION OF PSM EXON/INTRON JUNCTION SEQUENCES EXON 1 INTRON1 1F. Strand CGGCTTCCTCTTCGG (SEQ ID NO: 57) cggcttcctcttcggtaggggggcgcctcgcggag ...tatttttca (SEQ ID NO. 58) 1R strand...ataaaaagtCCCACCAAA (SEQ ID NO: 59) Exon 2 Intron 2 2F. strandACATCAAGAAGTTCT (SEQ ID NO: 60) acatcaagaagttct caagtaagtccatactcgaag(SEQ ID NO: 61) 2R. strand ...caagtggtcATTAAAATG (SEQ ID NO: 62) Exon 3Intron 3 3F. strand GAAGATGGAAATGAG (SEQ ID NO: 63) gaagatggaaatgaggtaaaatataaataaataaataa (SEQ ID NO: 64) Exon 4 Intron 4 4F. strandAAGGAATGCCAGAGG (SEQ ID NO: 65) aaggaatgccagagg taaaaacacagtgcaacaaa(SEQ ID NO: 66) 4R. strand agagttgTCCCGCTAGAT (SEQ ID NO: 67) Exon 5Intron 5 5F. strand CAGAGGAAATAAGGT (SEQ ID NO: 68) CAGAGGAAATAAGGTaggtaaaaattatctctttttt (SEQ ID NO: 69) ... gtgttttctAGGTTAAAAATG (SEQ IDNO: 70) 5R. strand ...cacttttgaTCCAATTT (SEQ ID NO: 71) Exon 6 Intron 66F. strand GTTACCCAGCAAATG (SEQ ID NO: 72) gttacccagcaatggtgaatgatcaatccttgaat (SEQ ID NO: 73) 6R. strand...aaaaaaagtCTTATACGAATA (SEQ ID NO: 74) Exon 7 Intron 7 7F. strandACAGAAGCTCCTAGA (SEQ ID NO: 75) acagaagctcctaga gtaagtttgtaagaaaccargg(SEQ ID NO: 76) 7R. strand aaacacaggttatcTTTTTACCCA (SEQ ID NO: 77) Exon8 Intron 8 8F. strand AAACTTTTCTACACA (SEQ ID NO: 78) aaacttttctacacagttaagagactatataaatttta (SEQ ID NO: 79) 8R. strand...aaacgtaatcaTTTTCAGTTCTAC (SEQ ID NO: 80) Exon 9 Intron 9 9F. strandAGCAGTGGAACCAG (SEQ ID NO: 81) agcagtggaaccag gtaaaggaatcgtttgctagca(SEQ ID NO: 82) ...tttctagatAGATATGTCATTC (SEQ ID NO: 83) 9R. strand...aaagaTCTGTCTATACAGTAA (SEQ ID NO: 84) Exon 10 Intron 10 10F. strandCTGAAAAAGGAAGG (SEQ ID NO: 85) ctgaaaaaggaagg taatacaaacaaatagcaagaa(SEQ ID NO: 86) Exon 11 Intron 11 11F. strand TGAGTGGGCAGAGG (SEQ ID NO:87) Agagg ttagttggtaatttgctataatata (SEQ ID NO: 88) Exon 13 Intron 1212R. strand GAGTGTAGTTTCCT (SEQ ID NO: 89) Gtagtttcctgaaaataagaaaagaatagat (SEQ ID NO: 90) Exon 14 Intron 13 13R. strandagggcttttcagct acacaaattaaaagaaaaaaag (SEQ ID NO: 92) Exon 14 Intron 1314F. strand GTGGCATGCCCAGG (SEQ ID NO: 93) gtggcatgcccaggtaaataaatgaatgaagtttcca (SEQ ID NO: 94) Exon 16 Intron 15 15R. strandAATTTGTTTGTTTCC (SEQ ID NO: 95) aatttgtttgtttcc tacagaaaaaacaacaaaaca(SEQ ID NO: 96) Exon 16 Intron 16 16F. strand CAGTGTATCATTTG (SEQ ID NO:97) cagtgtatcatttg gtatgttacccttcctttttcaaatt (SEQ ID NO: 98)TttcagATTCACTTTTTT (SEQ ID NO: 99) 16R. strand aaagtcTAAGTGAAAA (SEQ IDNO: 100) Exon 17 Intron 17 17F. strand TTTGACAAAAGCAA (SEQ ID NO: 101)ttttgacaaaagcaa gtatgttctacatatatgtgcatat (SEQ ID NO: 102) 17R. strand...aaagagtcGGGTTA (SEQ ID NO: 103) Exon 18 Intron 18 18F. strandGGCCTTTTTATAGG (SEQ ID NO: 104) ggcctttttatagg taaganaagaaaatatgactcct(SEQ ID NO: 105) 18R. strand ...aatagttgTGTAAACCC (SEQ ID NO: 106) Exon19 Intron 19 19F. strand GAATATTATATATA (SEQ ID NO: 107) gaatattatatatagttatgtgagtgtttatatatgtgtgt (SEQ ID NO: 108) Notes: F: Forward strand R:Reverse strand

1. A method of detecting expression of an alternatively spliced prostatespecific membrane antigen in a cell or tissue, which alternativelyspliced prostate specific membrane antigen consists essentially ofconsecutive amino acids, the amino acid sequence of which is set forthin SEQ ID NO:128 beginning with methionine at position number 58 andending with alanine at position number 750, the nucleic acid encodingthe alternatively spliced prostate specific membrane antigen having anintron splice site located between the G and T nucleotides at positions114 and 115, respectively, as set forth in SEQ ID NO:1, wherein a spliceat said site results in formation of an exon-exon junctioncharacteristic of said alternatively spliced prostate specific membraneantigen, said method comprising: (1) contacting mRNA obtained from thecell or tissue with a detectable nucleic acid of at least 15 nucleotidesin length which specifically hybridizes across said exon-exon junction;and (2) determining whether the detectable nucleic acid hybridizes tothe mRNA, wherein the presence of the detectable nucleic acid hybridizedto the mRNA indicates expression of alternatively spliced prostatespecific membrane antigen in the cell or tissue.
 2. The method of claim1, wherein the detectable nucleic acid is labeled with a detectablelabel.
 3. The method of claim 2, wherein the detectable label is aradioisotope or fluorescent dye.